Quality Control Solutions for Next-Gen Sequencing Workflows

Sample Quality Control in Next-Generation Sequencing Workflows Agilent Automated Electrophoresis Portfolio Application compendium - 2nd edition Introduction Throughout the updated edition of this application compendium, you will find various application notes and technical overviews providing you essential information for success in your nucleic acid sample quality assessment during next-generation sequencing (NGS) workflows. This collection of resources includes topics covering: – Why sample quality control is important in NGS workflows – How quality metrics help provide reliable assessment of sample integrity – How to analyze and assess quality of cell-free DNA (cfDNA), formalin-fixed paraffin-embedded (FFPE) DNA and RNA, genomic DNA (gDNA), total RNA, and NGS library samples – Which instrument is most suitable for different workflows and applications Recommended steps for quality control during NGS library preparation. Sample preparation Input sample QC Library preparation Intermediate sample QC Sequencing Target enrichment Final library QC Automated Electrophoresis Instruments to Support Your NGS Workflows Next-generation sequencing is an essential tool in molecular biology laboratories for the analysis of nucleic acid samples in numerous basic, translational, and clinical research settings. Quality control (QC) of input samples, at intermediate steps of the preparation process, and of the final libraries before sequencing, can help save time and resources by identifying samples that are of poor quality or of insufficient concentration to yield successful sequencing data. The Agilent automated electrophoresis portfolio provides several instruments for QC of various nucleic acid sample types to support your NGS workflow. Discover which Agilent automated electrophoresis solution fits your application. Select an instrument below for more information. Automated parallel capillary electrophoresis providing accurate and reliable quality assessment for different sample types with a broad range of kits. Automated pulsed-field capillary electrophoresis enabling exceptional QC for low concentration nucleic acid samples and high-molecular weight DNA. See how the Agilent Bioanalyzer system compares to our other automated electrophoresis instruments. Automated electrophoresis QC for various workflows for the analysis of size, quantity, and integrity of RNA and DNA input samples. Automation for Library Preparation and Target Enrichment Prepare high-quality, sequencing-ready libraries with more consistency and lab efficiency using automated solutions for library preparation and target enrichment. Choose between two automation platforms from Agilent, both of which support enzymatic fragmentation of DNA, reverse transcription for RNA, and bead cleanup. These automation platforms offer a Reagent Rental Program for SureSelect reagents to offset capital equipment expense. Simplified NGS workflow automation without sacrificing quality, in a small footprint. High-throughput NGS sample preparation robot for increased efficiency and maximized walkaway time. Compact benchtop platform providing an automated, completely walkaway NGS library preparation and target enrichment solution. NGS Products For Reliable Results Besides sample QC instrumentation and automation options, Agilent offers an extensive porfolio of NGS products that help you generate reliable data. Agilent SureSelect hybridization capture-based reagents include custom and catalog kits for library preparation and target enrichment to meet your sequencing needs. The Agilent Alissa Clinical Informatics platform enables innovative NGS and CGH data analysis to optimize workflows across technologies and applications. The platform seamlessly and efficiently integrates raw read alignment, variant annotation and classification, and reporting modules. See which products are best suited for your NGS-based research. Agilent TapeStation Systems The Agilent TapeStation systems offer scalable throughput and rapid results, making them the ideal solution for quality control of biological samples in NGS. While the higher throughput Agilent 4200 TapeStation system can analyze DNA and RNA samples from a 96-well plate, the smaller footprint Agilent 4150 TapeStation instrument is the lower throughput alternative for analyzing up to 16 samples per run. Both systems offer walk away operation with fully automated sample processing. No matter which system you choose, full assay compatibility is guaranteed as both instruments share the same ScreenTape consumables. ScreenTape details At-a-glance expiry date Individual sample lanes Zero carry-over Barcode Automated assay detection Buffer chamber Automatic sample loading Electrodes Individual sample separation Overview of Agilent ScreenTape device. Agilent Fragment Analyzer Systems The Agilent Fragment Analyzer systems use automated parallel capillary electrophoresis (CE) to provide reliable quality control (QC) of nucleic acids for various applications, including nextgeneration sequencing. The 5200, 5300, and 5400 Fragment Analyzer systems are used for low to high throughputs, breaking through analytic bottlenecks and streamlining nucleic acid analysis workflows to provide researchers with accurate and reliable results. Automated parallel capillary electrophoresis can analyze multiple samples at once without researcher intervention. The systems can house two different gel matrices enabling unattended and consecutive analysis of multiple RNA and DNA reagent kits. Overview of capillary electrophoresis process. Light Charged Coupled Device (CCD) Capillaries Sample or buffer tray Reservoir High voltage power supply Agilent Femto Pulse System The Agilent Femto Pulse system delivers unparalleled sensitivity and sizing of nucleic acids, enabling accurate and reliable sizing of gDNA smears and large fragments through 165 kb. The Femto Pulse system is a powerful and effective automated, pulsed-field capillary electrophoresis system with the ability to separate high-molecular weight (HMW) DNA through 165,000 bp. An optimized optical platform allows the system to easily achieve 10 times higher sensitivity for nucleic acid smears and 100 times higher for nucleic acid fragments compared to the other automated electrophoresis systems. The Femto Pulse was designed for the qualitative and quantitative analysis of nucleic acid sample with low concentrations or HMW DNA. Overview of capillary electrophoresis process. Agilent 2100 Bioanalyzer System For more than 20 years, molecular biologists have benefited from RNA and DNA analysis using the Agilent 2100 Bioanalyzer system. Starting in January 2024, the Bioanalyzer instrument will no longer be available for purchase, however, its corresponding kits and reagents will remain available. The new generation of Agilent automated electrophoresis instrumentation, including the TapeStation, Fragment Analyzer and Femto Pulse systems, deliver additional advanced quality metrics for the analysis of specialized sample types such as genomic DNA, cell-free DNA, and total RNA libraries. To see how sample analysis using the Bioanalyzer system compares to our other automated electrophoresis instruments, explore these resources: Comparison of DNA Sample QC for NGS Workflows with the Agilent Fragment Analyzer and Bioanalyzer Systems Comparison of Small DNA Fragment Analysis using the Agilent Bioanalyzer and Agilent Fragment Analyzer Systems Comparison of Small RNA Analysis using the Agilent Bioanalyzer and Agilent Fragment Analyzer Systems Quality Assessment of NGS Libraries using Agilent Automated Electrophoresis Systems Comparison of the Agilent 2100 Bioanalyzer and the Agilent Fragment Analyzer Systems for Analysis of Plant, Insect, and Bacterial RNA Comparison of DNA Assays Using the 4200 TapeStation System and 2100 Bioanalyzer System Performance Characteristics of the RNA and the High Sensitivity RNA ScreenTape Assays for the 4150 TapeStation System 01 02 03 04 05 06 07 Proven Quality Metrics for Multiple NGS Applications Quality metrics provide you with a reliable assessment of sample integrity so you can ensure your nucleic acids are sufficient for successful library preparation and sequencing results. Whether you are working with genomic DNA (gDNA), cell-free DNA (cfDNA), formalin-fixed paraffin-embedded (FFPE) DNA and RNA, or total RNA you can find reliable quality metrics suited to your application. Explore each of the metrics below and learn about their use in sample quality control with automated electrophoresis instruments. Bioanalyzer system Fragment Analyzer systems Femto Pulse system TapeStation systems FFPE RNA Total RNA gDNA/ FFPE DNA cfDNA NGS Quality Control for a Wide Range of Nucleic Acids Robust sample analysis is crucial throughout the NGS workflow for successful sequencing results. Whether you need to assess the integrity of your cell-free DNA (cfDNA), formalin-fixed paraffin-embedded (FFPE) DNA and RNA, genomic DNA (gDNA), total RNA, or NGS libraries, our automated electrophoresis instruments offer you support. Select your desired sample type to explore relevant application notes and technical overviews: High Throughput Genomic DNA Assessment by the Agilent 4200 TapeStation System Abstract When used with the Genomic DNA ScreenTape assay, the 4200 TapeStation system can separate and analyze genomic DNA (gDNA) from 200 to 60,000 bp. It provides an automated numerical assessment of gDNA quality, the DNA integrity number (DIN). The DIN is calculated using a scale from 1 to 10. A high DIN indicates highly intact gDNA, and a low DIN suggests a strongly degraded gDNA sample. The user-independent DIN is the ideal QC tool for next-generation sequencing (NGS) and array comparative genomic hybridization (aCGH) workflows. Three different mouse gDNA samples with different DNA integrity were analyzed using the 4200 TapeStation system. The TapeStation analysis software displays the results as an electropherogram, a gel image, and data table. The DIN value is automatically determined, and directly displayed under the individual lane of the gel image (A). The corresponding samples are shown in the electropherogram overlay (B). Technical overview 48,50 0 15,00 0 7,00 0 4,00 0 3,00 0 2,50 0 2,00 0 1,50 0 1,20 0 90 0 60 0 40 0 25 0 10 0 48,500 15,000 7,000 4,000 3,000 2,500 2,000 1,500 1,200 900 600 400 250 100 DIN 9. 8 [bp] DIN 5. 8 DIN 1. 8 0 20 0 40 0 60 0 80 0 1,000 1,200 1,400 1,600 Size (bp) Sample intensity (FU) Genomic DNA analysis Evaluating the Agilent 4200 TapeStation System for High Throughput Sequencing Quality Control Abstract To evaluate the difference of the DNA integrity number (DIN) between the 2200 and the 4200 TapeStation systems, two commercially available gDNA samples were analyzed at three different concentrations using the Genomic DNA ScreenTape assay. The DNA integrity number (DIN) is automatically determined and directly displayed below the individual lane of the gel image. A DIN is calculated on a scale from 1 to 10. A high DIN indicates highly intact gDNA, whereas a low DIN corresponds to a strongly degraded gDNA. Both gDNA samples were highly intact, as indicated by the determined DIN, which is displayed below the gel image. The bar chart illustrates that the DNA integrity analysis with the 4200 TapeStation system is highly comparable to that of the 2200 TapeStation system. The precision for the DNA integrity analysis for both systems is 4% for all tested samples and concentrations. Application Note no dil. DIN 9.1 DIN 9.5 DIN 9.5 DIN 8.7 DIN 8.6 DIN 9.4 DIN 9.4 DIN 9.5 DIN 9.7 DIN 8.8 DIN 8.6 DIN 9.6 [bp] 1:2 48,500 15,000 7,000 4,000 3,000 2,500 2,000 1,500 1,200 900 600 400 250 100 48,500 15,000 7,000 4,000 3,000 2,500 2,000 1,500 1,200 900 600 400 250 100 1:4 no dil. 1:2 1:4 [bp] Agilent 4200 TapeStation system Agilent 2200 TapeStation system gDNA sample A gDNA sample B no dil. 1:2 1:4 no dil. 1:2 1:4 gDNA sample A gDNA sample B 0 1 2 3 4 5 6 7 8 9 10 no dil. 1:2 1:4 no dil. 1:2 1:4 DIN Agilent 4200 TapeStation system Agilent 2200 TapeStation system gDNA sample A gDNA sample B Genomic DNA analysis Quality Control for Agilent SureSelect QXT WGS Library Preparation Abstract The Agilent SureSelect QXT WGS protocol requires high-quality DNA samples for optimal performance and precise quantification of the gDNA starting material. Serial quantification was carried out using the Qubit instrument and the dsDNA BR assay in accordance with the protocol. The same samples were analyzed on the 4200 TapeStation system with the Genomic DNA ScreenTape assay and the NanoDrop with six replicates on each instrument. Data from the 4200 TapeStation system, Qubit, and NanoDrop are presented in the figure, showing the applicability of the Genomic DNA ScreenTape assay in quantitating genomic DNA starting material. The quantification results of the TapeStation system correlate with the Qubit instrument. The measurement of genomic DNA with UV spectroscopy tends to overestimate the quantity due to other buffer components that may absorb in the UV spectrum. In addition, the Genomic DNA ScreenTape assay provides an objective assessment of sample integrity within the same QC step. Sample integrity is automatically determined by the DNA integrity number (DIN) calculation provided by the TapeStation analysis software. Application note 0 10 20 30 40 50 60 70 First dilution Second dilution Concentration (ng/µL) Qubit Agilent 4200 TapeStation Nanodrop 48,500 [bp] A1 DIN 9.2 DIN 7.9 DIN 8.3 B1 C1 15,000 7,000 4,000 3,000 2,500 2,000 1,500 1,200 900 600 400 250 100 Genomic DNA analysis GQN Quality Metrics with the Fragment Analyzer and Femto Pulse Systems Abstract Quality assessment of nucleic acids is critical to the success of many downstream applications, including next-generation sequencing (NGS). The Fragment Analyzer and Femto Pulse systems provide quick and easy assessment of genomic DNA (gDNA) quality and integrity with the genomic quality number (GQN). The GQN is commonly used for evaluating the input gDNA material for NGS library preparation. To prepare a successful library, the sample must be of the correct size and of sufficient quality for sequencing. The GQN threshold can be set by the user to reflect the size threshold necessary for their particular requirements. The GQN is given on a scale of 0 to 10, with a higher score indicating that more of the sample exceeds the user-defined threshold. In this example, the Femto Pulse system was used to report the average smear size (A) and GQN set at 30 kb (B) of several gDNA samples that had been sheared to various sizes. For NGS, quality metrics can aid decisions about library preparation, including which samples to use for input material, the number of PCR cycles, and the amount of library to use for enrichment. Application note Average smear size (bp) GQN Set at 30 kb 12,147 0 23,339 1.5 45,304 6.4 57,789 7.1 73,267 7.8 94,045 7.8 109,968 8.2 164,292 8.8 1 0 Size (bp) LM RF U 2,000 4,000 6,000 8,000 10,000 12,000 14,000 16,000 18,000 1,300 10,000 17,700 23,000 42,000 50,000 165,500 A B Genomic DNA analysis Assessment of Genomic DNA Quality with the Agilent 5200 Fragment Analyzer System Abstract The quality and concentration of genomic DNA (gDNA) starting material is crucial for successful downstream long-read and wholegenome sequencing. Quality analysis for gDNA with varying ranges of concentrations can be performed on the Fragment Analyzer systems with the Genomic DNA 50 kb kit and the HS Genomic DNA 50 kb kit. The Genomic DNA 50 kb kit offers a concentration range of 25 to 250 ng/μL input gDNA, while the HS Genomic DNA 50 kb kit has a lower concentration range of 0.3 to 12 ng/μL input gDNA for low concentrated samples. Genomic DNA from cotton, E. coli, and human (Coriell) were compared on both kits with the FA 12-Capillary Array Short, 33 cm (short array), and FA 12-Capillary Array Ultrashort, 22 cm (ultrashort array). On both kits, the short and ultrashort arrays demonstrated consistent sizing for the three sample types. The ultrashort array offers the convenience of shortened run times while providing comparable gDNA sizing, concentration, and genomic quality number (GQN) compared to the short array with both kits. Application note 1 0 A Short array LM RFU Size (bp) 500 1,000 1,500 2,000 2,500 3,000 3,500 4,000 4,500 5,000 5,500 6,000 75 200 400 600 800 1,000 3,000 6,000 10,000 15,000 48,500 1,500 Cotton (DNF-467) Cotton (DNF-468) Coriell (DNF-467) Coriell (DNF-468) E. coli (DNF-467) E. coli (DNF-468) 1 0 B Ultrashort array LM RFU Size (bp) 1,000 2,000 3,000 4,000 5,000 6,000 7,000 8,000 9,000 75 200 400 600 800 1,000 3,000 6,000 10,000 15,000 48,500 1,500 Cotton (DNF-467) Cotton (DNF-468) Coriell (DNF-467) Coriell (DNF-468) E. coli (DNF-467) E. coli (DNF-468) 1 C DNF-467 LM RFU Size (bp) 75 200 400 600 800 1,000 3,000 6,000 10,000 15,000 48,500 1,500 Cotton (short) Cotton (ultrashort) Coriell (short) Coriell (ultrashort) E. coli (short) E. coli (ultrashort) Cotton (short) Cotton (ultrashort) Coriell (short) Coriell (ultrashort) E. coli (short) E. coli (ultrashort) 1 D DNF-468 LM RFU Size (bp) 0 1,000 2,000 3,000 4,000 5,000 6,000 7,000 8,000 75 200 400 600 800 1,000 3,000 6,000 10,000 15,000 48,500 1,500 0 500 1,000 1,500 2,000 2,500 3,000 3,500 4,000 4,500 5,000 5,500 6,000 Genomic DNA analysis Genomic DNA Sizing and Quality Control on the Agilent Femto Pulse System Abstract Large-insert library preparation relies upon multiple quality control steps. Typically, overnight pulsed-field gel electrophoresis (PFGE) separations are used to assess gDNA over 50 kb in size. The Agilent Femto Pulse system is the only instrument on the market capable of replacing PFGE with fast, automated assessment of high-molecular weight (HMW) gDNA, saving time and money in the preparation of largeinsert libraries. The Genomic DNA 165 kb kit offers two pulsed-field capillary electrophoresis separation methods. The gDNA 165 kb method is a 70-minute method recommended for gDNA under 80 kb. The extended Genomic DNA 165 kb method provides enhanced separation and sizing for larger samples in 3.5 hours. Large gDNA separated with the fast method displayed a sharp, compact peak around 165 kb (A). The same sample analyzed with the extended method resulted in a broader smear representing the entire sizing range of the sample (B). This application note shows comparable sizing of four gDNA samples separated on the Femto Pulse and traditional PFGE. It also demonstrates consistent sizing of samples throughout a dilution series and discusses the application of genomic quality number (GQN) provided by the Femto Pulse system. Application note 1 4,000 A Size (bp) LM 179,342 bp RFU 5,000 6,000 7,000 8,000 9,000 10,000 1,300 10,000 17,700 21,000 23,000 42,000 50,000 165,500 1 4,000 B Size (bp) LM 157,440 bp RFU 4,200 4,400 4,600 4,800 5,000 5,200 5,400 5,600 1,300 10,000 17,700 21,000 23,000 42,000 50,000 165,500 Genomic DNA analysis Comparison of Different Methods to Isolate HMW DNA from Bacteria for Nanopore Sequencing Abstract The first step in many sequencing experiments is to isolate nucleic acids from a specimen. As long-read sequencing technologies have advanced, many commercial companies now offer kits specific to the extraction of highmolecular weight (HMW) genomic DNA (gDNA). However, the size and quality of the gDNA can vary greatly depending upon the extraction method used. For sequencing facilities, this can affect the type of sequencing that is performed, making knowledge of sample integrity crucial to a successful experiment. In this application note, researchers at the NGS Competence Center Tuebingen (NCCT) used the Agilent Femto Pulse system to analyze HMW gDNA from five different commercially available extraction kits. The size of the input material was then compared to the sequenced read lengths to demonstrate the impact that the isolation method can have on sample size and sequencing results. An example from one of the five extraction kits is shown in the figure. Data generated from the Femto Pulse demonstrated that the extraction method used resulted in a large peak with some smearing to the left of the peak, indicating the presence of smaller fragments within the sample (A). The distribution of the sample was confirmed with Nanopore sequencing results, which showed a large amount of fragments of smaller sizes (B), but also some as big as 500 kb (C). Application note 39201 37500 35000 32500 30000 Size (bp) RFU A 27500 22500 20000 17500 17500 15000 10000 7500 5000 1857 1 1300 10000 17700 21000 23000 42000 50000 165500 25000 LM 136625 # of reads (logarithmic scale) Fragment length (bp) 0.9 0.6 0.3 0.0 1,000 10,000 100,000 B Read length 9e–05 6e–05 3e–05 0e–00 100 kb 100 kb 200 kb 200 kb 300 kb 300 kb 400 kb 400 kb 500 kb # of reads sequenced 500 kb C Genomic DNA analysis Comparison of Agilent Femto Pulse System Sizing with Long-Read Sequencing Read Length Abstract Long-read sequencing results can be maximized by loading only long fragments onto the sequencer, thereby eliminating any issues with preferential sequencing of smaller fragments. This can be achieved through size selection, to exclude the portion of the sample below a specified threshold. Sheared samples, with and without size selection, were analyzed with the Agilent Femto Pulse system, and then sequenced on the Oxford Nanopore Technologies MinION. The size distribution of the samples reported by the Femto Pulse was similar to the histogram of the sequencing read lengths. Shown here is an example of a 15 kb Blue Pippin (15 kb BP) size-selected gDNA analyzed on the Femto Pulse (B). The sample was sequenced with the Oxford Nanopore Technologies MinION, and histograms of the read lengths were generated from the data with NanoPlot (D). Size selection eliminated the small fragments present in the sample, resulting in similar sizes between the Femto Pulse average size and the mean sequencing results. The Femto Pulse confirmed effective size selection, which was further confirmed by the sequencing results. Application note 10586 10000 9500 9000 8500 8000 7500 7000 6500 6000 5500 4762 RF U 1 1300 Size (bp) 10000 17700 21000 23000 42000 165500 50000 LM 9746 11470 10000 10500 11000 9500 9000 8500 8000 7500 7000 6500 6000 5500 5000 4450 RF U 1 1300 Size (bp)10000 17700 21000 23000 42000 165500 50000 LM 12743 40000000 35000000 30000000 25000000 Number of bases 20000000 15000000 10000000 5000000 0 0 20000 40000 Read length 60000 80000 100000 Weighted Histogram of read lengths Weighted Histogram of read lengths 40000000 Number of bases 30000000 20000000 10000000 0 0 20000 40000 Read length 60000 80000 100000 120000 140000 20 kb g-TUBE sheared 15 kb BP 25000 20000 15000 10000e (bp) Average size of input gDNA compared to mean sequencing read length B D Genomic DNA analysis Comparison of Constant- and Pulsed-Field Electrophoresis Technologies for Analysis of High Molecular Weight and Large DNA Fragments Abstract Choosing the appropriate electrophoresis technology is critical to achieving successful downstream results. Constant- and pulsed-field electrophoresis technologies are both designed to separate DNA based on size. Constant-field technology is traditionally thought to be best suited for separation of DNA smears that are less than 20 kb, based on previous studies. Beyond 20 kb, DNA smears that are analyzed using constant-field technology may be impacted by the compression of samples, which occurs due to the stacking of DNA within the gel. Pulsed-field technology is capable of separating DNA smears with no known compression effects, as seen in constant-field technology. Agilent offers two capillary electrophoresis instruments that use these different technologies: the Agilent Fragment Analyzer systems use constantfield technology, while the Agilent Femto Pulse system uses both pulsed- and constant-field technologies. As shown in the figures, the data presented in this application note indicates that while both the Fragment Analyzer and Femto Pulse can be used for analysis, the Femto Pulse provides more accurate sizing for high molecular weight (HMW) DNA, genomic DNA (gDNA), and sheared samples, both within the range of 10 to 20 kb, and above. Application note A C B Size (bp) 38000 36000 34000 32000 30000 28000 26000 24000 22000 20000 18000 16000 14000 12000 10000 8000 6000 4000 2000 0 Fragment Analyzer gDNA 50 kb kit Femto Pulse gDNA 165 kb kit Sample A Sample B Sample C Expected sample size A) 10 kb B) 15 kb C) 20 kb Genomic DNA analysis Impact of gDNA Integrity on the Outcome of DNA Methylation Studies Abstract DNA methylation is the most widely studied modification involved in epigenetics. In mammalian cells, DNA methylation mainly involves the transfer of a methyl group from S-adenosyl methionine to the carbon 5 position of a cytosine residue to produce 5-methylcytosine. DNA methylation is mainly implicated in the repression of transcriptional activity. This application note focuses on 5-methylcytosine (5-mC) DNA methylation and its detection. By far, the most commonly used method for DNA methylation analysis is bisulfite sequencing due to its high-resolution detection when combined with sequencing. Advances in next-generation sequencing made it possible to perform bisulfite sequencing at a genomewide scale. The DNA methylation analysis based on reduced representation bisulfite sequencing, a high-throughput technique, allows analysis on a single nucleotide level. This method is based on the enrichment of genome regions with high CpG content (sites within the genome where a cytosine is next to a guanine) using a combination of restriction enzymes and bisulfite sequencing and is applicable to any species with a reference genome. Shown here is a comparison of the DNA methylation levels for overlapping CgP sites of four FFPE samples. DIN values ranged from 4 to 7.3, to a fresh frozen sample with a DIN value of 8 using Hexbin plots. Application note DIN 4.0 1.0 0.8 0.6 0.4 0.2 0 0 0.2 0.4 0.6 DIN 4.0 DIN 8.0 0.8 1.0 5.4 4.8 4.2 3.6 3.0 log10(N) 2.4 1.8 1.2 0.6 0 1.0 0.8 0.6 0.4 0.2 0 0 0.2 0.4 0.6 DIN 7.1 DIN 8.0 0.8 1.0 5.4 4.8 4.2 3.6 3.0 log10(N) 2.4 1.8 1.2 0.6 0 DIN 7.1 1.0 0.8 0.6 0.4 0.2 0 0 0.2 0.4 0.6 DIN 5.8 DIN 8.0 0.8 1.0 5.4 4.8 4.2 3.6 3.0 log10(N) 2.4 1.8 1.2 0.6 0 1.0 0.8 0.6 0.4 0.2 0 0 0.2 0.4 0.6 DIN 7.3 DIN 8.0 0.8 1.0 5.6 4.8 4.0 3.2 log10(N) 2.4 1.6 0.8 0 DIN 5.8 DIN 7.3 DIN 4.0 1.0 0.8 0.6 0.4 0.2 0 0 0.2 0.4 0.6 DIN 4.0 DIN 8.0 0.8 1.0 5.4 4.8 4.2 3.6 3.0 log10(N) 2.4 1.8 1.2 0.6 0 1.0 0.8 0.6 0.4 0.2 0 0 0.2 0.4 0.6 DIN 7.1 DIN 8.0 0.8 1.0 5.4 4.8 4.2 3.6 3.0 log10(N) 2.4 1.8 1.2 0.6 0 DIN 7.1 1.0 0.8 0.6 0.4 0.2 0 0 0.2 0.4 0.6 DIN 5.8 DIN 8.0 0.8 1.0 5.4 4.8 4.2 3.6 3.0 log10(N) 2.4 1.8 1.2 0.6 0 1.0 0.8 0.6 0.4 0.2 0 0 0.2 0.4 0.6 DIN 7.3 DIN 8.0 0.8 1.0 5.6 4.8 4.0 3.2 log10(N) 2.4 1.6 0.8 0 DIN 5.8 DIN 7.3 Genomic DNA analysis Comparison of Small RNA Analysis using the Agilent Bioanalyzer and Agilent Fragment Analyzer Abstract Analysis of small RNA samples can help with optimization of small RNA isolation and purification protocols and ensure successful downstream applications such as small RNA next-generation sequencing (NGS) (RNASeq), miRNA microarrays, and qRT-PCR. The systems of the automated electrophoresis portfolio from Agilent have been well established for the analysis of total RNA, and uniquely offer quality analysis of small RNAs with either the Agilent 2100 Bioanalyzer system, or the Agilent Fragment Analyzer systems. Both instruments use kits that focus on small RNA and miRNA quality control, concentrating on a narrow range of 200 nt and below, and allowing for high-separation resolution of small RNAs. In this technical overview, samples are compared across both the Bioanalyzer and the Fragment Analyzer Small RNA kits to demonstrate the equivalency of the systems for analyzing small RNAs.The figure shows a commercially available total RNA that was further analyzed on A) the Agilent Bioanalyzer Small RNA kit and B) the Agilent Fragment Analyzer Small RNA kit to allow for higher separation resolution of the small RNA portion of the sample below 200 nt. Technical overview A B Total RNA analysis Evaluating the Agilent 4200 TapeStation System for High Throughput Sequencing Quality Control Abstract As RNA is highly sensitive to degradation, quality control is essential, and usually includes RNA integrity analysis and quantification. The electropherogram overlay shown here represents different dilutions of liver total RNA analyzed on the 4200 TapeStation system with the RNA ScreenTape assay (A). The RNA integrity number equivalent (RINe ), indicating RNA integrity, is automatically determined by the TapeStation software. The RINe is calculated on a scale from 1 to 10. A high RINe indicates highly intact total RNA, whereas a low RINe corresponds to a strongly degraded sample. The bar chart (B) illustrates that RNA integrity analysis is highly reproducible and independent of the analyzed concentration for both TapeStation systems. In comparison to the 2200 TapeStation system, the 4200 TapeStation system further reduces manual operating time, enabling the analysis of 96 samples without physical intervention. This is a primary requirement for highthroughput NGS labs. Application note 0 1 2 3 4 5 6 7 8 9 10 1:8 1:20 1:35 RI Ne Agilent RNA ScreenTape assay Agilent 4200 TapeStation system Agilent 2200 TapeStation system Marker Liver total RNA 1:8 1:20 1:35 0 0 2 4 6 7 10 12 14 16 ×103 25 200 500 1,000 Sample intensity (FU) Size [nt] 2,000 4,000 6,000 A B Total RNA analysis Quality Control in Illumina Sequencing Workflows Using the TapeStation System Abstract RNA is sensitive to degradation due to the ubiquitous presence of RNase and its more fragile single-stranded structure. Therefore, monitoring the integrity of starting material is indispensable, and processing a reference sample as positive control throughout the library preparation and sequencing is highly advisable. With the RNA ScreenTape assay, the RNA integrity number equivalent (RINe ) delivers an objective assessment of the integrity of RNA starting material. RINe is a proven equivalent to the widely accepted quality metric RIN. The fragmentation conditions of RNA‑seq protocols used by the sequencing core facility are optimized for high-quality RNA; more precisely, a RINe of 8.0 or higher is recommended for successful library preparation. The figure shows two samples close to this threshold, one passing (A) and one failing (B) the quality requirement. The use of degraded RNA can result in low yield, over-representation of 3’ ends of the RNA molecules, or failure of the protocol. Application note Lower 18S 28S 0 1 2 3 4 5 ×103 Sample intensity (normalized FU) A 25 200 500 1,000 2,000 6,000 Size (bp) Lower 18S 28S 0 1 2 3 4 5 ×103 Sample intensity (normalized FU) B 25 200 500 1,000 2,000 6,000 Size (bp) Total RNA analysis Quality Analysis of Eukaryotic Total RNA with the Agilent 5200 Fragment Analyzer System Abstract The RNA quality number (RQN) is a user-independent quality metric for easy evaluation of total RNA quality. Total RNA quality is a constant concern because of how easily RNA degrades due to heat, RNase exposure, and improper handling. The 5200 Fragment Analyzer system and RQN metric were used to analyze universal mouse reference total RNA degradation over time. Electropherogram (A) and digital gel images (B) allow for the total RNA profiles to be compared at different time points of heat degradation. As shown, the 18S ribosomal peak becomes smaller, while the 28S ribosomal peak completely disappears as the sample is further degraded. This degradation strongly correlates with a decrease in RQN (C), allowing for easy assessment of RNA quality. Application note 15 0 A B Size (nt) LM RF U 200 15 200 500 1,000 1,500 2,000 3,000 4,000 6,000 Size (nt) 400 600 800 1,000 1,200 1,400 F1 F2 F3 F4 F5 F6 F7 F8 F9 F10 F11 F12 200 500 1,000 1,500 2,000 3,000 4,000 6,000 0246 8 10 12 14 16 18 20 0 Minutes at 70 °C RQ N 1 2 3 4 5 6 7 8 9 C 10 15 0 A A Size (nt) LM RF U 200 15 200 500 1,000 1,500 2,000 3,000 4,000 6,000 Size (nt) 400 600 800 1,000 1,200 1,400 F1 F2 F3 F4 F5 F6 F7 F8 F9 F10 F11 F12 200 500 1,000 1,500 2,000 3,000 4,000 6,000 02468 10 12 14 16 18 20 0 Minutes at 70 °C RQ N 1 2 3 4 5 6 7 8 9 C 10 Total RNA analysis Performance Characteristics of the RNA and High Sensitivity RNA ScreenTape Assays for the 4150 TapeStation System Abstract RNA serves as input material within various gene expression analysis techniques like RNA-Seq, microarray, and RT-qPCR. RNA is very sensitive to degradation and its integrity critically affects the success of the downstream applications. Quality control of RNA input material is crucial for ensuring high-quality results. The 4150 TapeStation system can be used with the RNA ScreenTape assay to separate and analyze RNA, providing an automated numerical assessment of RNA quality, the RNA integrity number equivalent (RINe ). The RINe is calculated using a scale from 1 to 10. A high RINe indicates highly intact RNA, and a low RINe suggests a strongly degraded RNA sample. The RINe was demonstrated to be equivalent to RIN from the 2100 Bioanalyzer system. The user-independent RINe is the ideal QC tool for next-generation sequencing (NGS) workflows. Four rat kidney RNA samples with different degradation stages were analyzed using the 4150 TapeStation system. The 4150 TapeStation software displays the results as an electropherogram, a gel image, and data table. The RINe value is automatically determined, and directly displayed under the individual lane of the gel image (A). (B) shows the corresponding electropherogram overlay. Comparison of the RINe from the 4150 and 4200 TapeStation systems was evaluated and found to be equivalent. Technical overview A B [nt] A1 RINe 3.2 RINe 3.1 RINe 3.2 RINe 5.0 RINe 5.0 RINe 5.0 RINe 6.7 RINe 6.8 RINe 6.9 RINe 8.6 RINe 8.6 RINe 8.5 B1 C1 D1 E1 F1 G1 H1 A2 B2 C2 D2 25 200 500 1,000 2,000 4,000 6,000 0 5 10 15 20 25 30 Size (nt) Sample intensity (normalized FU) 25 200 500 1,000 2,000 4,000 6,000 ×103 Sample 4 Sample 3 Sample 2 Sample 1 Total RNA analysis Monitoring Library Preparation for Next-Generation Sequencing in Systems Biology Omics Analysis Abstract RNA samples are often subject to degradation by RNases, chemical, or other environmental impacts. Therefore, RNA integrity analysis is a crucial step before any downstream application like NGS. The RINe metric from the TapeStation software is equivalent to the RIN metric provided by the RNA assays of the Bioanalyzer system. The comparative analysis of 30 RNA samples extracted from mouse cancer tissue verified that RIN and RINe highly correlate, with a slope of 0.997 and a goodness-of-fit (R2 ) of 97.9% (A). The same samples were quantified fluorometrically and results were compared with the RNA ScreenTape assay. Both assays resulted in a similar total RNA concentration of samples, with a slope of 1.017 and a R2 of 97.9%, showing high correlation (B). Two degraded RNA samples were treated as outliers. Overall, the RNA ScreenTape assay provides a very useful tool in determining integrity and quantity of RNA starting material for sequencing in a single analysis step. Application note y = 1.0172x + 16.555 R² = 0.9791 0 100 200 300 400 500 600 700 800 0 100 200 300 400 500 600 700 Concentration (ng/µL) Qubit RNA HS assay Concentration (ng/µL) RNA ScreenTape assay y = 0.9973x – 0.1922 R² = 0.9788 0 1 2 3 4 5 6 7 8 9 10 012345678 9 10 RINe (4150 TapeStation system) RIN (2100 Bioanalyzer system) A B Total RNA analysis Comparison of RIN and RQN for the Agilent 2100 Bioanalyzer and the Fragment Analyzer Systems Abstract The Bioanalyzer instrument is well established for providing a reliable, automated RNA integrity number (RIN). The RIN provides an objective assessment of RNA integrity. The Fragment Analyzer offers a userindependent quality metric, the RNA quality number (RQN), for easy evaluation of total RNA quality. Both the RIN and RQN consider the entire electropherogram, with scoring from 1 to 10, where 10 indicates the highest possible RNA quality and 1 completely degraded RNA. Eukaryotic samples with a varying degree of RNA integrity, from completely intact, to mildly and strongly degraded, were compared on the Bioanalyzer and Fragment Analyzer instruments. Both the standard sensitivity (A) and High Sensitivity RNA (B) kits on both instruments provided comparable RIN and RQN scores throughout the degradation series. This is demonstrated with the slope and R 2 value close to 1. This technical overview also shows a strong correlation between the RIN and RQN for prokaryotic E. coli RNA samples. Technical overview SS RNA Kits Eukaryotic RNA Bioanalyzer Fragment Analyzer 12.0 10.0 8.0 6.0 4.0 2.0 0.0 0 2 4 6 8 10 12 y = 0.9574x + 0.4957 R² = 0.9628 Bioanalyzer Fragment Analyzer HS RNA Kits Eukaryotic RNA 0 02468 10 12 2 4 6 8 10 12 y = 0.9875x + 0.0423 R2 = 0.9746 AB Total RNA analysis Comparison of RNA Quality Analysis with the Qubit RNA IQ Assay and Agilent Automated Electrophoresis Systems Abstract RNA integrity can be determined using electrophoresis methods that separate the sample based on size, allowing for the distinction of the ribosomal peaks, small RNA, and any degradation products. The Agilent 2100 Bioanalyzer system and the Agilent Fragment Analyzer systems are used for RNA quality control (QC), providing information about both the quantity and the quality of a given sample. The systems both generate an objective, reliable quality metric score for each sample analyzed. These scores are known as the RNA integrity number (RIN) for the Bioanalyzer, and the RNA quality number (RQN) for the Fragment Analyzers. Each score is based on a scale from 1 to 10, with 1 identifying significantly degraded samples and 10 identifying high-quality, intact RNA specimens. The RNA Integrity and Quality (IQ) Assay kit for the Qubit 4.0 fluorometer from Thermo Fisher Scientific also offers an RNA quality score, representative of the ratio of small and large RNAs present in the sample. In this technical overview, the RIN and RQN scores from the Agilent automated electrophoresis instruments are compared to the Qubit RNA IQ score across a series of RNA reference samples from multiple species. Technical overview Human Heart RNA Quality Scores 10.0 C) Comparison of quality scores 8.0 6.0 4.0 2.0 0.0 RIN (Bioanalyzer) RQN (Fragment Analyzer) RNA IQ (Qubit) A) Agilent Bioanalyzer System B) Agilent Fragment Analyzer System 43000 40000 37500 35000 32500 30000 27500 25000 22500 20000 17500 15000 12500 10000 7500 5000 2500 -1500 Size (nt) RFU 15 200 500 1000 1500 2000 3000 4000 6000 [FU] 40 35 30 25 20 15 10 5 0 25 200 500 1000 2000 4000 [nt] Red: intact Blue: mildly degraded Green: severely degraded Black: intact Red: mildly degraded Blue: severely degraded Human Heart RNA Quality Scores 10.0 C) Comparison of quality scores 8.0 6.0 4.0 2.0 0.0 RIN (Bioanalyzer) RQN (Fragment Analyzer) RNA IQ (Qubit) Human heart intact Human heart mild degradation Human heart severe degradation A) Agilent Bioanalyzer System B) Agilent Fragment Analyzer System 43000 40000 37500 35000 32500 30000 27500 25000 22500 20000 17500 15000 12500 10000 7500 5000 2500 -1500 Size (nt) RFU 15 200 500 1000 1500 2000 3000 4000 6000 [FU] 40 35 30 25 20 15 10 5 0 25 200 500 1000 2000 4000 [nt] Red: intact Blue: mildly degraded Green: severely degraded Black: intact Red: mildly degraded Blue: severely degraded Total RNA analysis Comparison of the Agilent 2100 Bioanalyzer and the Agilent Fragment Analyzer Systems for Analysis of Plant, Insect, and Bacterial RNA Abstract Evaluating RNA integrity prior to downstream analysis saves time, effort, and resources by ensuring that only samples of sufficient quality are used. To aid in objectively determining the integrity of a sample, quality metrics can be assigned that help to grade the quality of the RNA, independent of user bias. The Agilent 2100 Bioanalyzer and 5200 Fragment Analyzer systems each provide reliable, convenient analysis of samples. Each system also provides a quality metric for RNA samples, called the RNA integrity number (RIN) and the RNA quality number (RQN), respectively. The RIN and RQN assign a score from 1 to 10 for a given RNA sample. A score of 1 indicates severely degraded RNA, while a score of 10 indicates highly intact RNA. In this technical overview, different total RNA samples were evaluated to compare the quality metrics between the Bioanalyzer and Fragment Analyzer systems. Plant, insect, and bacterial total RNA samples were analyzed using both the high- and standard sensitivity kits for each system, to provide a detailed comparison of the RIN and RQN. The electropherograms here show corn analyzed on the (A) Agilent 2100 Bioanalyzer system and (B) Agilent 5200 Fragment Analyzer system. Technical overview A B 15 200 500 1000 1500 2000 3000 4000 7000 6500 6000 5500 5000 450 0 4000 3500 3000 2500 2000 1500 1133 5 RFU 7500 8000 8500 9000 9500 10000 1050 0 1100 0 Size (nt) 16 0 14 0 [FU] 20 40 60 80 10 0 12 00 Total RNA analysis Use of the Agilent 4200 TapeStation System for Sample Quality Control in the Whole Exome Sequencing Workflow at the German Cancer Research Center (DKFZ) Abstract To determine if samples were suitable for NGS library preparation, a quality control (QC) assessment was performed at the beginning for a batch of 88 genomic DNA (gDNA) samples from FFPE tumor tissue by the German Cancer Research Center (DKFZ) High Throughput Sequencing Unit. This initial QC includes quantification and analysis with a 4200 TapeStation system and the Genomic DNA assay to determine DNA quality, based on the DNA integrity number (DIN). Both figures show a representative subset of samples analyzed on the 4200 TapeStation with the Genomic DNA ScreenTape assay. gDNA samples extracted from FPPE material often have low DNA integrity but can still be sufficiently intact for whole-exome sequencing library preparation protocols and successful sequencing. Application note B 500 400 300 200 100 Sample intensity [FU] 0 Size (bp) A [bp] 48,500 15,000 7,000 4,000 3,000 2,500 2,000 1,500 1,200 900 600 400 250 100 DIN 2.9 DIN 4.9 DIN 3.9 DIN 3.1 DIN 2.6 DIN 1.5 DIN 4.4 DIN 4.4 DIN 3.2 DIN 3.6 DIN 2.5 DIN 2.3 DIN 3.3 DIN 3.7 DIN 3.5 B4 C4 D4 E4 F4 G4 H4 A5 B5 C5 D5 E5 F5 G5 H5 15,000 48,500 1,200 1,500 2,000 2,500 3,000 4,000 7,000 900 600 400 250 100 FFPE analysis The DNA Integrity Number (DIN) Provided by the Genomic DNA ScreenTape Assay Allows for Streamlining of NGS on FFPE Tissue Samples Abstract Sequencing of genomic DNA (gDNA) from FFPE archived tissue can be challenging, as the obtained material is often of variable quality. This study demonstrates that the DNA integrity number (DIN) obtained by the quality control of gDNA using the Agilent Genomic DNA ScreenTape assay has allowed for a pronounced saving of sequencing and sample preparation overhead. Out of a total of 751 FFPE samples, a subset of 197 were tested for a correlation of various NGS parameters against the DIN. A correlation was identified between DIN and the key parameters of on-target rate and coverage at 10x. A QC threshold of ≥ 3 DIN was therefore set, which consequently excluded 65% (n = 488) of the total sample set and saved a significant amount of time and effort. The right panel is the gel image, and the left panel is the electropherogram. Application note 0 100 Size (bp) 100 250 400 600 900 1,200 1,500 2,000 2,500 3,000 4,000 7,000 15,000 48,500 200 300 400 500 600 bp 48,500 DIN 4.1 DIN 5.2 DIN 5.0 DIN 2.2 DIN 2.9 DIN 1.9 15,000 7,000 4,000 3,000 2,500 2,000 1,500 1,200 900 600 400 250 100 Sample intensity (FU) 1 2 3 4 5 6 FFPE analysis FFPE Sample Quality for the MGISEQ-2000 Sequencing Platform with the Agilent TapeStation System Abstract Formalin-fixed paraffin embedding (FFPE) is one of the more common ways to preserve clinical samples, and FFPE tumor samples serve as valuable study materials for clinical and translational medicine research. To ensure successful and reliable sequencing of FFPE-derived DNA, it is necessary to conduct quality control (QC) of both the initial DNA, as shown here, as well as fragmented samples at key points during next-generation sequencing (NGS) library construction. In this application note, the Agilent 4150 TapeStation system was used to conduct QC of the entire library construction process for five FFPE DNA samples used on the MGI Tech MGISEQ2000RS sequencing platform. High-quality sequencing results were achieved by stepwise QC throughout the library preparation process, including the initial sample, pre- and post-PCR steps, and the final library, helping to ensure high-quality sequencing. Application note No. Type of Tissue DIN Concentration (ng/µL) Main Peak (bp) FFPE-1 Cervical 2.9 28.6 1,919 FFPE-2 Colorectal 4.5 84.8 2,241 FFPE-3 Thyroid 2.6 42.4 731 FFPE-4 Breast 2.2 10.2 574 Control Standards 7.9 32.4 24,312 1200 A1 B1 C1 D1 E1 H1 Composite 1000 800 600 Sample Intensity [Normalized FU] 400 200 0 100 250 400 600 900 1200 1500 2000 2500 3000 4000 7000 15000 48500 Size [bp] A1) gDNA kit ladder (100 bp - 48.5 kb) B1 to E1) FFPE samples 1 to 4 H1) FFPE DNA standard FFPE analysis DV200 Evaluation with RNA ScreenTape Assays Abstract The DV200 quality metric represents the percentage of RNA fragments above 200 nucleotides and shows a high correlation to the precapture library yield of RNA samples originating from formalin-fixed paraffin-embedded (FFPE) tissue. The RNA and High Sensitivity RNA ScreenTape assays enable fast and easy analysis of FFPE RNA samples, with the TapeStation software displaying DV200 results after region setup as percentage of total. DV200 region setup can be automated for repeated FFPE RNA sample analysis and all region data can be exported and reported. Both the RNA ScreenTape assay and High Sensitivity RNA ScreenTape assay yielded highly comparable results. Technical overview FFPE analysis Detection of Contaminating High Molecular Weight DNA with the Cell-Free DNA ScreenTape Assay Abstract Cell-free DNA represents a challenge for next-generation sequencing (NGS) workflows due to the low yield, complex fragmentation pattern, and possibility of contaminating high molecular weight (HMW) DNA. To appropriately assess the quality of cfDNA, it is important to visualize the fragmentation pattern and any degradation or contamination within the sample. Assessment of the cfDNA fragments can be performed using Agilent automated electrophoresis instruments, including the Bioanalyzer and TapeStation systems, as shown in the figure. However, the presence of HMW DNA contamination, which can lead to misrepresentation of the total sample concentration and negatively affect NGS library yield and sequencing results, can overlap with the Upper Marker used by the Bioanalyzer, impacting assessment of the total sample. Alternately, the Cell-free DNA ScreenTape assay for the TapeStation systems reliably visualizes HMW DNA contamination and calculates a %cfDNA quality score for objective assessment of the amount of cfDNA in the sample compared to any HMW DNA contamination. This technical overview compares the analysis of cfDNA contaminated with HMW DNA between the Bioanalyzer and TapeStation systems. Technical overview LM UM 35 100 150 200 300 400 500 600 1000 2000 10380 [bp] Size [bp] [FU] 160 140 120 100 80 60 40 20 -20 0 Mononucleosome Dinucleosome Trinucleosome HMW region Mononucleosome Dinucleosome Trinucleosome HMW DNA 35 50 75 100 150 200 300 400 500 600 700 1000 0 100 200 300 400 Sample Intensity [Normalized FU] Lower 50 7 cfDNA region 00 A. Agilent 2100 Bioanalyzer system B. Agilent 4200 TapeStation system Cell-free DNA analysis Performance Characteristics of the Agilent Cell-free DNA ScreenTape Assay Abstract To demonstrate the concentration independence of the %cfDNA metric, a dilution series of a reference cfDNA sample (n=24), covering the entire concentration range of the assay, was analyzed. The average %cfDNA value was 85.0% ± 1.0 with a minimum value of 83.7% and maximum value of 86.6%. The %cfDNA results and the electropherogram profiles were consistent over the entire concentration range of the assay. In the figure, each concentration is overlaid with three replicates to demonstrate the consistency and precision of the Cell-free DNA ScreenTape assay. These results show that the %cfDNA quality metric provided by the Cell-free DNA ScreenTape assay is highly accurate and precise, and that the percentage is independent of the sample concentration. Technical overview Size (bp) 1500 4500 pg/µL 2000 pg/µL 800 pg/µL 400 pg/µL 200 pg/µL 80 pg/µL 1000 500 0 35 50 75 10 0 15 0 20 0 30 0 40 0 50 0 60 0 70 0 1000 Sample Intensity [Normalized FU] Cell-free DNA analysis cfDNA Separated on the Agilent Femto Pulse System Abstract The Femto Pulse system offers unparalleled sensitivity for the analysis of low concentrated cfDNA samples. In addition, the system’s high resolution provided allows samples with multiple fragments to have complete separation between peaks. cfDNA samples can differ in the number of fragments present, but the mono- and dinucleosome fragments are usually both present. cfDNA analyzed on the Femto Pulse with the Ultra Sensitivity NGS kit displayed six fragments (A). In the dilution series (250 to 7.8 pg/μL), the first three cfDNA fragments were completely separated at all concentrations, with the fourth fragment easily distinguishable down to 15.6 pg/μL. The fifth and sixth fragment peaks were less apparent below 31.3 pg/μL. A seventh fragment peak was observed only in the highly concentrated samples, 62.5 pg/μL and higher. Sizing remained consistent for all six peaks throughout the concentration range they were visible for (B). Application note 1 0 Size (bp) LM UM RFU 200 400 600 800 1,000 1,200 1,400 1,600 1,800 100 200 300 400 500 600 700 800 900 1,000 1,200 1,500 2,000 3,000 6,000 1 0 cfDNA fragment s Size (bp) 200 400 600 800 1,000 1,200 1,400 2 3 4 5 6 174 375 Sizing 574 783 963 1,170 AB 250 pg/µL 125 pg/µL 62.5 pg/µL 31.3 pg/µL 15.6 pg/µL 7.8 pg/µL Cell-free DNA analysis Quality Control of Cell-free DNA Samples Analyzed with Next-Generation Sequencing Abstract Cell-free DNA (cfDNA) has become an important input material for NGS as a result of the noninvasive collection methods from blood and urine. However, analysis of cfDNA can introduce challenges due to its low concentration and possible contamination from highmolecular weight DNA, both necessitating reliable quality control. The Agilent 4200 TapeStation system is a vital quality control (QC) tool in NGS workflows. The TapeStation systems and Cellfree DNA ScreenTape assay provide a %cfDNA quality metric for determining the quality of input cfDNA for downstream processes. As part of the German Cancer Research Center (Deutsches Krebsforschungszentrum), the Sample Processing Lab collaborated with the Genomics and Proteomics Core Facility to track 13 cfDNA samples with the TapeStation system, from initial QC with the %cfDNA metric (see Figure), through the NGS workflow, to the final sequencing results. All 13 samples reported a %cfDNA greater than 83%, with successful library preparation and sequencing metrics. Application note 0 35 50 75 100 150 200 300 400 500 600 700 1000 Size [bp] Sample Intensity [Normalized FU] 1000 2000 3000 4000 Lower %cfDNA = 97% Mono- A. Sample no. 5 Di50 700 0 35 50 75 100 150 200 300 400 500 600 700 1000 Size [bp] Sample Intensity [Normalized FU] 20 40 60 100 80 120 140 Lower %cfDNA = 83% MonoB. Sample no. 8 Di- Tri50 700 cfDNA samples 0 1000 Size [bp] Sample Intensity [Normalized FU] 100 200 300 400 500 Lower Upper Average region size 353 bp A. Sample no. 5 25 50 100 200 300 400 500 700 1500 Average region size 436 bp 100 0 25 50 100 200 300 400 500 700 1000 1500 Size [bp] Sample Intensity [Normalized FU] 200 300 400 600 500 700 Lower Lower B. Sample no. 8 Final NGS libraries Cell-free DNA analysis Use of the Agilent 4200 TapeStation System for Sample Quality Control in the Whole Exome Sequencing Workflow at the German Cancer Research Center (DKFZ) Abstract The 4200 TapeStation system was used for quality control of the final NGS libraries. These were expected to be sized between 250 and 350 bp with a minimum concentration of 2 ng/μL. In a gel view of 15 samples, lane B5 and D6 show negative controls (A). B shows an example of an electropherogram of one sample. The distribution of the concentration for all 80 samples plus eight controls is illustrated in C. The two positive controls are shown as green symbols. The red lines indicate the recommended concentration threshold (2 ng/μL). The maximum peak size for all 80 samples plus eight controls is displayed in D. The two positive controls are shown as green symbols. The red lines indicate the recommend size range (250 to 350 bp).The analysis of the final NGS libraries with the 4200 TapeStation system confirmed successful DNA library preparation for all 80 samples and the six positive control samples. Application note 0 5 10 15 20 25 30 0 20 40 60 80 100 Concentration (ng/µL) 0 50 100 150 200 250 300 350 0 20 40 60 80 100 Size (bp) Sample Sample A B C D [bp] 1,500 1,000 700 500 400 300 200 50 100 L3 (L) G4 H4 A5 B5 C5 D5 E5 F5 G5 H5 A6 B6 C6 D6 E6 25 1,000 1,500 700 500 400 300 200 25 50 100 Sample intensity [FU] 0 1,000 2,000 3,000 4,000 5,000 Size (bp) Lower 268 Upper NGS library analysis Evaluating the Agilent 4200 TapeStation System for High Throughput Sequencing Quality Control Abstract Quality control of NGS libraries is key to the success of any sequencing run. The D1000 ScreenTape and High Sensitivity D1000 ScreenTape assays can be used for quality control, providing DNA sizing, and quantification. The bar chart shows the sizing results of a sample in 3 dilutions analyzed with the High Sensitivity D1000 ScreenTape assay on both the 2200 and 4200 TapeStation platforms. DNA concentration determined with the 4200 TapeStation system is plotted against the concentration measured with the 2200 TapeStation system for the High Sensitivity D1000 ScreenTape assay. The data demonstrates that results obtained with the Agilent High Sensitivity D1000 assay using both TapeStation systems are directly comparable and highly reproducible. Application note Marker Marker DNA sample C 1:25 1:30 1:36 0 0 100 25 50 100 200 300 400 500 700 1,000 1,500 200 300 400 500 Sample intensity (FU) Size [bp] 0 100 1:25 1:30 1:36 200 300 400 500 600 700 Size [bp] DNA sample C DNA sample D y = 1.0344x R2 = 0.9832 500 600 500 Agilent 2200 TapeStation concentration [pg/µL] Agilent 4200 TapeStation system Agilent 2200 TapeStation system DNA sample C Agilent 4200 TapeStation concentration [pg/µL] 600 700 800 900 1,000 700 800 900 1,000 A B C NGS library analysis Quality Control for Agilent SureSelect QXT WGS Library Preparation Abstract For multiplex sequencing, SureSelectQXT whole-genome libraries are pooled so that each index-tagged sample is present in equimolar amounts in the final pool. The 4200 TapeStation and 2100 Bioanalyzer systems provide molarity and quantification data along with the sizing information in the region table of the software. For each library generated by various gDNA input amounts, the molarity was plotted in a graph comparing both systems. The data summarized in the table demonstrates that sizing and quantification of amplified libraries with the High Sensitivity D5000 ScreenTape assay match the results of the High Sensitivity DNA assay of the 2100 Bioanalyzer system. Application note 0 500 1,000 1,500 2,000 2,500 20 ng Male DNA 50 ng Male DNA 80 ng Male DNA Molarity (pmol/L) Agilent 4200 TapeStation Agilent 2100 Bioanalyzer Starting material Average size (bp) Region molarity (pmol/L) Agilent 4200 TapeStation System Agilent 2100 Bioanalyzer System Agilent 4200 TapeStation System Agilent 2100 Bioanalyzer System 20 ng mean 519 533 850 817 % CV 1.2 2.8 5.4 0.6 50 ng mean 849 868 1513 1473 % CV 0.6 1.4 1.7 4.5 80 ng mean 1065 1157 2073 2097 % CV 3.8 1.1 4.3 5.5 NGS library analysis A 0 25 100 300 500 700 1,000 1,500 Size (bp) 100 500 400 300 200 Lower Upper Sample intensity (normalized FU) Sample intensity (normalized FU) 35 100 200 300 400 600 1,000 2,000 10,380 Size (bp) 35 150 300 500 1,000 10,380 Size (bp) 0 50 100 150 200 0 50 100 150 250 200 Sample intensity (normalized FU) Sample intensity (normalized FU) C 0 25 100 300 500 700 1,000 1,500 Size (bp) 500 2,500 2,000 1,500 1,000 Lower Upper 50 200 400 B D HaloPlex HS HaloPlex HS HaloPlex HaloPlex Sample Quality Control in Agilent NGS Solutions Abstract HaloPlex and HaloPlex HS target enrichment technology uses an amplicon-based approach. The final libraries of HaloPlex and HaloPlex HS workflows show a profile with a characteristic smear in the range of 175 to 625 bp, (A) and (B), and 190 to 545 bp respectively (C) and (D). The appearance of the profile may vary due to specific library designs and the overall quality of the input material. The electropherogram should be checked for the presence of artefactual peaks with sizes less than 150 bp, as these are related to primer dimers that can cluster and consume sequencing capacity. If the primer dimer peak is greater than 10% of the total product, an additional cleanup step with AMPure beads is recommended. Application note NGS library analysis Quality Control in Illumina Sequencing Workflows Using the TapeStation System Abstract NGS target enrichment enables a detailed analysis of specific regions to identify causal genetic variants of complex conditions. The SureSelect XT protocol is designed to create libraries with enriched targeted regions of the genome for sequencing with Illumina platforms. The two intermediate QC steps include evaluation of a smear size after shearing (A) and before capturing (B). These steps can be carried out using the D1000 ScreenTape assay. The expected size range of the maximum peak of sheared DNA is 150 to 200 bp. For precapture library, a larger maximum peak size of 225 to 275 bp is expected due to adapter ligation. The last QC step qualifies the final library before pooling (C). Another size shift is expected as a result of adding index sequences. The peak maximum of the final library is expected to be between 250 and 350 bp. A minimum concentration of 2 ng/μL is expected for successfully generated final libraries. Application note 100 25 50 Lower 264 Upper 200 300 400 500 700 1,000 1,500 0 1 2 3 4 5 6 7 8 9 Sample intensity (normalized FU) Size (bp) ×103 100 25 50 Lower 173 Upper 200 300 400 500 700 1,000 1,500 0 1 2 3 4 5 ×103 Sample intensity (normalized FU) Size (bp) 100 25 50 Lower 286 Upper 200 300 400 500 700 1,000 1,500 0 1 2 3 4 5 Sample intensity (normalized FU) Size (bp) ×103 A C B NGS library analysis Comparison of the Agilent HS Small Fragment Kit and Agilent HS NGS Fragment Kit on the Fragment Analyzer Systems Abstract The quality of NGS libraries is crucial to successful sequencing results. The Fragment Analyzer systems offer easy analysis of sheared genomic DNA (gDNA) and libraries with the HS NGS Fragment kit (1-6000 bp) and the HS Small Fragment kit. The HS NGS Fragment kit (C and D) analyzes larger smears and fragments up to 6,000 bp, while the HS Small Fragment kit (A and B) focuses on smaller sizes up to 1,500 bp. The FA 12-Capillary Array Ultrashort 22 cm decreases run time by 10 to 20 minutes compared to the standard FA 12-Capillary Array Short, 33 cm. The size and concentration of several DNA smears were compared between both kits and the short and ultrashort arrays. Library sizing and quantification remained consistent between the short (A and C) and ultrashort (B and D) arrays and the two kits. The HS Small Fragment kit and the HS NGS Fragment kit can be used interchangeably for sizing and quantification of NGS libraries, as long as the sample fits within the sizing range of the kit. Application note 0 LM 50 100 150 200 250 300 400 500 600 700 800 900 1,000 UM 1,500 500 1,000 1,500 2,000 2,500 3,000 3,500 4,000 4,500 A 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 Time (min) RFU 153 0 LM UM 200 400 600 800 1,000 1,200 1,400 B RFU 1 Size (bp) 100 50 150 200 250 300 400 500 600 700 800 1,000 1,500 0 LM 153 UM 500 1,000 1,500 2,000 2,500 3,000 3,500 4,000 C RFU 1 Size (bp) 100 50 150 200 250 300 400 500 600 700 800 1,000 1,500 0 LM 326 UM 200 400 600 800 1,000 1,200 1,400 1,600 1,800 E RFU 1 Size (bp) 100 200 300 400 500 600 700 800 1,000 1,500 3,000 6,000 0 0.4 1,0.8 1,200 1,600 2,000 2,400 2,800 RFU 1 100 200 300 400 500 600 700 800 1,000 1,500 3,000 6,000 LM 325 UM F Size (bp) 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 Time (min) 0 LM UM 1,000 6,000 2,000 3,000 4,000 5,000 6,000 D RFU 100 200 300 400 500 600 700 800 900 1,000 1,200 1,500 2,000 3,000 HS Small kit ladder ultrashort array HS NGS kit ladder ultrashort array HS Small kit short array Sample #1 HS NGS kit short array Sample #3 HS Small kit ultrashort array Sample #1 HS NGS kit ultrashort array Sample #3 A. HS Small Fragment kit, short array, sample #1 C. HS NGS Fragment kit, short array, sample #3 B. HS Small Fragment kit, ultrashort array, sample #1 D. HS NGS Fragment kit, ultrashort array, sample #3 NGS library analysis Monitoring Library Preparation for Next-Generation Sequencing in Systems Biology Omics Analysis Abstract Frequently, the first step of a library preparation protocol is the fragmentation of gDNA by shearing with an ultrasonicator. Optimal shearing in NGS workflows can be verified by evaluating the size distribution and electropherogram pattern of fragmented DNA samples using the 2100 Bioanalyzer (A) and the 4150 TapeStation systems (B) with the DNA 1000 kit and D1000 ScreenTape assays, respectively. Electropherograms of sheared DNA in this example display an even size distribution with no undesirable shouldering. The fragmented DNA samples show a maximum peak size between 260 and 310 bp on both systems, verifying optimal shearing (C). The size of the sample at this QC step can be compared to the size of sample after adapter ligation in the library preparation workflow, at which point a shift in size is expected. Overall sizing results of the 2100 Bioanalyzer and the 4150 TapeStation systems correlated highly with an average deviation of 2.2% for all 30 samples analyzed. Application note A C 0 20 40 60 80 100 120 Size (bp) FU 15 50 100 150 200 300 400 500 700 1,500 B 2 4 6 8 10 12 14 16 0 Size (bp) 25 50 100 200 300 400 500 700 1,500 1,000 Sample intensity (normalized FU) ×103 Lower Upper 1,000 100 0 50 100 150 200 250 300 350 400 123456789 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 Average size (bp) Sample number DNA 1000 assay D1000 ScreenTape assay NGS library analysis Quality Control of NGS Libraries with Daisy Chains Abstract The Bioanalyzer, TapeStation, and Fragment Analyzer systems with dedicated high sensitivity assays allow unambiguous detection of daisy chains and accurate sizing of the target library peak. Two KAPA HyperPlus libraries with different levels of amplification were analyzed on the Bioanalyzer (A), TapeStation (B), and Fragment Analyzer (C) systems using the High Sensitivity DNA kit, High Sensitivity D5000 ScreenTape assay, and HS Small Fragment kit respectively. As shown in the overlays, both libraries (blue – library 1, red – library 2) contained the desired library peak with a pronounced secondary peak in library 2. The excessive amplification of library 2 resulted in the formation of daisy chains, which were observed as an additional higher molecular weight peak in all electropherograms. The daisy chains migrated slower through the gel matrix and were easily detected by all Agilent automated electrophoresis instruments using the respective assays. In this application note, we provide a recommendation for which assay to employ to reliably visualize daisy chain products in next-generation sequencing libraries. Furthermore, we emphasize the consistency between the instruments and reproducibility of analysis confirmed by results on a series of double dilutions. Application note 300 35 100 200 [FU] 250 200 150 100 50 0 300 400 500 700 2000 10380 [bp] 8000 7000 6000 5000 4000 3000 2000 1000 0 15 Sample Intensity [Normalized FU] 100 250 400 600 1000 1500 2500 3500 5000 10000 Size [bp] 2800 2600 3000 3200 3400 2400 2200 1800 14002000 RF 1600 U A B C 300 35 100 200 [FU] 250 200 150 100 50 0 300 400 500 700 2000 10380 [bp] 8000 7000 6000 5000 4000 3000 2000 1000 0 15 Sample Intensity [Normalized FU] 100 250 400 600 1000 1500 2500 3500 5000 10000 Size [bp] 2800 2600 3000 3200 3400 2400 2200 1800 1400 2000 RF 1600 U 1200 800 1000 600 200 400 20 1 50 100 150 200 250 300 400 500 600 700 800 900 1000 1500 Size [bp] A B C 300 35 100 200 [FU] 250 200 150 100 50 0 300 400 500 700 2000 10380 [bp] 8000 7000 6000 5000 4000 3000 2000 1000 0 15 Sample Intensity [Normalized FU] 100 250 400 600 1000 1500 2500 3500 5000 10000 Size [bp] 2800 2600 3000 3200 3400 2400 2200 1800 1400 2000 RF 1600 U 1200 800 1000 600 200 400 20 1 50 100 150 200 250 300 400 500 600 700 800 900 1000 1500 Size [bp] A B C NGS library analysis Quality Control of Magnis SureSelect XT HS Workflows with Agilent Automated Electrophoresis Solutions Abstract The Magnis NGS Prep system is an automated NGS library preparation solution for the SureSelect XT HS system. It addresses challenges of manual library preparation, such as hands-on time, expertise, optimization, and validation for diverse applications. Performing QC steps and quantification on the starting material, the materials derived from intermediate steps (optional), and the final library is beneficial in ensuring reliability and overall success of the sequencing data. QC steps can be performed with the automated electrophoresis portfolio of instruments, including the Bioanalyzer, TapeStation, and Fragment Analyzer systems. gDNA was assessed with the Genomic DNA ScreenTape assay on the 4150 TapeStation system for overall integrity and size in high-quality gDNA, and mildly, moderately, and highly degraded FFPE samples (A). This assay applies a quality score, the DNA integrity number (DIN), to each sample. The score is used to optimize the fragmentation step and determine the amount of input DNA to be used in library preparation. Post-capture libraries were assessed on all three platforms and displayed similar sizing (B). Application note B Size in bp NA 18507 (10X) Average Size Post-Shear Library Average Size Pre-Capture Library Average Size of Post-Capture Library HD799 HD799 NA 18507 HD798 HD803 HD803 4150 TapeStation 2100 Bioanalyzer 5200 Fragment Analyzer NA 18507 HD798 HD799 HD803 200 150 100 50 0 400 300 200 100 0 400 300 200 100 0 Size in bp NA 18507 (10X) Average Size Post-Shear Library Average Size Pre-Capture Library Average Size of Post-Capture Library HD799 HD799 NA 18507 HD798 HD803 HD803 4150 TapeStation 2100 Bioanalyzer 5200 Fragment Analyzer NA 18507 HD798 HD799 HD803 200 150 100 50 0 400 300 200 100 0 400 300 200 100 0 A NA18507 HD803 HD799 HD798 Sample Intensity (Normalized FU) Size 100 250 400 600 900 1200 1500 2000 2500 3000 4000 7000 [bp] 15000 48500 200 400 600 800 1000 0 NGS library analysis Comparison of DNA Sample QC for NGS Workflows with the Agilent Fragment Analyzer and Bioanalyzer Systems Abstract In this technical overview, precapture and final libraries were compared on both the Agilent Fragment Analyzer and Bioanalyzer systems with their corresponding DNA assays to demonstrate data equivalency across platforms. The DNA assays selected have similar analytical specifications, allowing for seamless comparison between the instruments. Both systems support standard-sensitivity (SS) and high-sensitivity (HS) kits, covering a wide range of sample concentrations that occur at different QC checkpoints throughout library preparation. Starting DNA material can vary greatly in concentration, depending on whether its source is from fresh material or ancient samples. The HS kits are ideal for conservation of low-concentration samples, and can be used at all recommended QC checkpoints. Alternatively, the SS kit has the advantage of analyzing high-concentration samples while eliminating time-consuming dilution steps. Having the option of both SS and HS kits simplifies the library preparation workflow. The SS and HS kits are compared in this technical overview. Technical overview [FU] 250 200 150 100 50 35 100 150 200 300 400 500 500 1000 2000 10380 [bp] 0 B. Bioanalyzer NGS final library no. 22 Region 1 10447 10000 9500 9000 8500 8000 7500 7000 6500 RFU 6000 5500 5000 4500 4000 3500 3000 2423 1 100 200 300 400 500 600 700 800 900 1000 1200 1500 2000 3000 6000 Size (bp) A. Fragment Analyzer NGS final library no. 22 420 bp Adapter dimer LM UM LM UM Adapter dimer 413 bp A. Fragment Analyzer NGS final library no. 22 B. Bioanalyzer NGS final library no. 22 NGS library analysis Preventive Control of Sequencing Through the Insert with the Agilent 5200 Fragment Analyzer System Abstract The Agilent Fragment Analyzer systems, together with the Agilent NGS Fragment kits and the companion Agilent ProSize data analysis software, offer a well-established solution for sample quality control (QC) in next-generation sequencing (NGS) workflows. This application note expands the capabilities of the ProSize smear analysis, and demonstrates how its functionality can be further applied to avert a specific sequencing issue called sequencing through the insert. This data is then introduced into the sequencing read and, in some cases, can increase the background noise, decreasing the quality of the overall sequencing run. The percent total value of the ProSize software allows a user to estimate the percentage of the library that will be sequenced through the insert based on the planned sequencing run method and electrophoretic profile of the library. Easy and comprehensive smear analysis enables the user to determine an optimal read length, and thereby minimize the number of unwanted bases present in the sequencing reads, saving expensive reagents and time for auxiliary data processing. Application note 2698 LM 2400 UM 2200 2000 1800 1600 1400 1200 1000 800 600 400 200 -107 1 100 200 300 400 500 600 700 800 900 1000 1200 1500 2000 3000 6000 Size (bp) RFU B 2698 LM 2400 UM 2200 2000 1800 1600 1400 1200 1000 800 600 400 200 -107 1 100 200 300 400 500 600 700 800 900 1000 1200 1500 2000 3000 6000 Size (bp) RFU C 2698 LM 2400 UM 2200 2000 1800 1600 1400 1200 1000 800 600 400 200 -107 1 100 200 300 400 500 600 700 800 900 1000 1200 1500 2000 3000 6000 Size (bp) RFU A ID Range ng/ µ L % Total nmole/L Avg. Size %CV A2: S1 - 2 ng/ µ L 110 bp to 1350 bp 2.3296 98.7 11.5454 332 23.30 220 bp to 1350 bp 2.2339 94.6 10.8748 338 21.67 286 bp to 1350 bp 1.7412 73.7 7.9547 360 18.82 NGS library analysis Quality Assessment of NGS Libraries using Agilent Automated Electrophoresis Systems Abstract The Agilent automated electrophoresis instruments, including the Bioanalyzer, Fragment Analyzer, and TapeStation systems, are well-suited for QC of NGS libraries, providing high-quality quantification, qualification, and sizing of nucleic acids to allow for confident assessment of samples throughout the NGS workflow. Each instrument offers specific benefits suited to meet a variety of individual laboratory needs, such as throughput, sensitivity, speed, and resolution. To demonstrate the equivalency of the systems to each other, NGS libraries were compared across each instrument. Each instrument offers a versatile assay portfolio that covers broad sizing and concentration ranges. The DNA analysis kits chosen for analysis of the NGS libraries in this technical overview have similar specifications, with a sizing range well-suited for the NGS smear, allowing for comparison between the instruments. This technical overview highlights the capability of each system to accurately size and quantify NGS libraries. Technical overview Average Conc. (pg/µL) %CV %error Instrument and Kit Library 1 Library 2 Library 1 Library 2 Library 1 Library 2 Bioanalyzer High Sensitivity DNA 469 464 3.95 2.45 17.20 17.74 Fragment Analyzer High Sensitivity NGS 487 479 10.86 14.41 13.99 15.13 Fragment Analyzer High Sensitivity Small Fragment 482 491 14.56 7.81 14.84 12.91 TapeStation High Sensitivity D1000 533 549 7.91 7.05 5.77 2.60 A. B. 0 100 200 300 400 500 600 700 Library 1 Library 2 Concentration (pg/µl) Quantification Comparison Bioanalyzer High Sensitivity DNA Fragment Analyzer High Sensitivity NGS Fragment Analyzer High Sensitivity Small Fragment TapeStation High Sensitivity D1000 Average Size (bp) %CV Instrument and Kit Library 1 Library 2 Library 1 Library 2 Bioanalyzer High Sensitivity DNA 431 470 0.35 0.43 Fragment Analyzer High Sensitivity NGS 419 448 0.96 0.22 Fragment Analyzer High Sensitivity Small Fragment 429 457 0.54 0.25 TapeStation High Sensitivity D1000 423 446 1.54 0.22 0 50 100 150 200 250 300 350 400 450 500 Library 1 Library 2 Size (bp) Sizing Comparison Bioanalyzer High Sensitivity DNA Fragment Analyzer High Sensitivity NGS Fragment Analyzer High Sensitivity Small Fragment TapeStation High Sensitivity D1000 A. B. NGS library analysis Detection of Adapter Dimers in NGS Libraries with the Agilent Fragment Analyzer and TapeStation Systems Abstract During the process of library preparation, DNA fragments are ligated to a known sequence, or adapter. These adapters allow the DNA fragments to bind to the flow cell during the sequencing run. Even small amounts of adapter dimers in a library can contribute to detrimental sequencing results. The presence of adapter dimers can affect the accuracy of library quantification, leading to suboptimal flow cell loading and reduced clustering efficiency. Smaller fragments, such as dimers, are also preferentially sequenced over larger fragments, so even low levels of adapter dimers could contribute to issues such as reduced output, lower diversity, and decreased genome coverage. While the optimal amount of adapter dimer in a library is zero, there are thresholds of adapter dimer in a library that may be present and still generate satisfactory sequencing results. Thus, it is highly recommended to minimize and remove adapter dimers from the library before sequencing. This technical overview highlights the sensitivity and resolution of the Agilent Fragment Analyzer and TapeStation systems to detect adapter dimers even lower than the recommended threshold of 0.5% and deliver an accurate assessment of the percentage of adapter dimer in the library. Technical overview 0.5% Fragment Fragment Analyzer HS NGS Kit TapeStation HS D5000 ScreenTape Assay Expected Adapter Dimer Fragment Size (bp) % (Conc.) Fragment Size (bp) % Integrated Area 5% 153 4.2% 124 4.7% 1% 154 0.9% 138 1.3% 0.5% 153 0.5% 138 0.8% 0.1% 154 0.1% Not detected Not detected Table 2. Reported size and percentage of adapter dimer present within an NGS library analyzed on the Agilent 5200 Fragment Analyzer system and the Agilent 4200 TapeStation system. 0.5% Fragment NGS library analysis Want to Explore Even More Resources? Performing quality control of your nucleic acid samples with Agilent automated electrophoresis solutions can help advance your next-generation sequencing workflows while conserving valuable time and resources. Browse all of our automated electrophoresis solutions at: www.agilent.com/en/product/automated-electrophoresis For additional information on NGS sample quality control solutions from Agilent, visit us at: www.agilent.com/en/solutions/genomics-applications-solutions/sample-QC/ngs Buy online: www.agilent.com/chem/store U.S. and Canada 1-800-227-9770 agilent_inquiries@agilent.com Europe info_agilent@agilent.com Asia Pacific inquiry_Isca@agilent.com For Research Use Only. Not for use in diagnostic procedures. PR7001-1876 © Agilent Technologies, Inc. 2024 Published in the USA, January 1, 2024 5994-6990EN