A Guide to Sample Preparation for Next-Generation Sequencing

1 How To Guide A Guide to Sample Preparation for Next-Generation Sequencing Aditi Verma, PhD Next-generation sequencing (NGS) has become an indispensable tool for discovery, diagnosis, prognosis, forensics, precision medicine and more. NGS involves executing massively parallel sequencing of DNA or RNA in a fast, efficient, cost-effective and high-throughput manner.1 The NGS technique is considerably more sensitive than its predecessor, Sanger sequencing.2 The increased sensitivity that comes with NGS also implies that any compromise with the sample preparation quality can easily skew the results. Some common issues that result from poor sample preparation techniques include the insufficient number and quality of reads, non-replicability of results and contamination with nucleic acid sequences from non-target tissue or organisms. These issues often come to light after the sequencing run has been completed, when a substantial amount of time and resources have already been spent on generating the data. Further, given the high costs involved in sequencing, it is generally not economical to repeat NGS experiments. Therefore, to ensure that the data obtained after sequencing is reliable and replicable, it is very important that the sample preparation is performed with utmost care. The sample preparation depends on the intended NGS application and the source tissue. The nucleic acids sequenced in NGS are DNA and RNA, and some applications include whole genome sequencing, whole exome sequencing, RNA sequencing, DNA methylation sequencing and single-cell sequencing. DNA, RNA or single-cell suspensions can be obtained from different types of freshly collected blood or biopsy tissue specimens from the organisms of study. Other sources include formalin fixed paraffin embedded tissue and extracts from cell and tissue culture. Sample preparation involves obtaining the tissue or cells of interest, isolating the target nucleic acid (DNA or RNA), fragmentation and library preparation. These libraries are then loaded into the sequencing machine where the DNA or RNA is sequenced based on the sequencing application. Although the choice of nucleic acid and the sample source varies from one experiment to another, there are a few important guidelines that are common to most sample preparation methods. A GUIDE TO SAMPLE PREPARATION FOR NEXT-GENERATION SEQUENCING 2 How To Guide 1. Experiment design Proper planning is crucial to a successful NGS experiment. The scientific hypothesis that is being addressed has to be laid out clearly before one begins the sample preparation. For example, if you wish to identify single nucleotide polymorphisms that may be associated with a certain disease, whole genome sequencing is most appropriate. You also need to determine the type of nucleic acid that is appropriate for your target NGS application. For example, if you wish to study the expression of all genes in your target tissue, RNA sequencing would be ideal to provide the necessary transcriptomic information. RNA sequencing also provides information on the levels of gene expression from exonic regions as well as the expression of protein isoforms that result from splicing. However, if you wish to study the disease-causing mutations in protein-coding exonic regions of DNA, you should perform whole exome sequencing with DNA as your sample. Further, the experiment needs to be designed with appropriate positive and negative controls. If you intend to compare the sequencing results from a test case against a control, replicates from each set need to be included in the same sequencing run. The number of replicates also has to be determined before starting the experiment. NGS experiment results often have huge batch variations from one run to the next; though these can be accounted for to some extent at the analysis stage, these are not ideal, and it is best to design the experiment in a way that batch effects can be reduced to a minimum. 2. Tissue/specimen handling The procedure of specimen acquisition, processing and handling can greatly affect the yield and quality of the isolated nucleic acid, which in turn affects the accuracy, reliability and consistency of the experiment.3 Specimen acquisition procedures need to be standardized so that the outcome does not vary from one specimen handler to the next. For example, if a single-cell sequencing experiment requires the specimen to be microdissected from a brain section, the dissection should be optimized to ensure that all replicates have been dissected from the same brain tissue region and there is no contamination from nearby regions, which might skew the results by introducing unwanted neuronal cell types. Specimen acquisition and processing should be performed in an environment that ensures that contamination of the specimen can be avoided. This is especially important during RNA isolation, as RNA is extremely prone to degradation due to the abundance of ribonucleases in the environment. Specimen handling temperatures also contribute to the quality of the nucleic acid and should be appropriately maintained. For example, flash-frozen tissue degrades rapidly when at room temperature and the handling time needs to be minimized in order to obtain good-quality RNA.4 Further, when working with autopsy tissue, the post-mortem interval can considerably affect the results of the experiment and thus, you need to maintain consistent post-mortem intervals for all the samples in the experiment. Post-mortem intervals have been shown to influence the quality of RNA isolated.5 A GUIDE TO SAMPLE PREPARATION FOR NEXT-GENERATION SEQUENCING 3 How To Guide 3. Isolation of nucleic acids The next step is the isolation of DNA/RNA. Here, it is important to estimate the amount of the final product that will be required for the sequencing run. This will help you to decide how much of the specimen tissue/cell pellet needs to be used as starting material for nucleic acid preparation. The nucleic acid preparation method is generally chosen based on the amount of nucleic acid required and the amount and nature of the available source tissue. For example, when RNA is to be isolated from microgram quantities of autopsy brain tissue, a very sensitive RNA preparation kit that is suited for RNA isolation from extremely small amounts of lipid-rich tissue can be selected. Maintaining proper labeling of the storage vials throughout the isolation process will ensure that the samples retain their identities. Further, once the DNA/RNA has been isolated, the yield should be estimated, and sample aliquots should be prepared to avoid contamination and multiple freeze–thaw cycles due to excessive handling of the same sample vial. For example, if you have estimated that you will need 800 nanograms of RNA for further processing and you have isolated 3 micrograms of RNA, it would be ideal to prepare three aliquots of 1 microgram (keeping a little extra to account for pipetting errors) for each sample. The samples need to be stored properly at low temperatures until the next step is performed. While DNA samples can be stored at -20°C, RNA samples are stored at -80°C. 4. Quality control of isolated nucleic acids In addition to estimating the yield of the DNA/RNA, the quality of the isolated nucleic acid also needs to be checked before proceeding to fragmentation and library preparation. Spectrophotometric absorbance readings can be obtained at wavelengths of 260 nm and 280 nm for DNA samples and at 260 nm and 230 nm for RNA samples. A 260/280 value (ratio of absorbances at 260 and 280 nm) of 1.80–2.00 indicates favorable DNA quality. Similarly, a 260/230 ratio of 2.00–2.20 indicates optimal RNA quality. DNA/RNA samples with lower values should not be considered for downstream processing. These samples can be purified further. The integrity of RNA samples is further quantified by estimating their RNA integrity number (RIN). This number ranges from 1 to 10, with 10 being the highest quality of RNA. It is general practice to not consider samples below a RIN of 5 for sequencing. 5. Library preparation Library preparation is considered an important step before sequencing and includes the fragmentation of DNA/RNA samples, the addition of adapter sequences that will allow the sequences to adhere to the flow cell for sequencing and the addition of index sequences that enable multiplexing and identification of samples during analysis. This step, therefore, needs to be performed using a kit that prepares the libraries to be compatible with the sequencer. Further, the library preparation method can be chosen in a manner to ensure that libraries can be prepared from poor quality or low yields of DNA/RNA samples. A GUIDE TO SAMPLE PREPARATION FOR NEXT-GENERATION SEQUENCING 4 How To Guide 6. Single-cell sequencing sample preparation Single-cell sequencing helps you to understand cellular heterogeneity. The sample in this case is a cell suspension that is loaded onto a microfluidic device where individual cells are mixed with barcoded beads. Thus, nucleic acids from single cells become barcoded and cell type heterogeneity in gene expression can be analyzed upon sequencing. To prepare a good quality cell suspension, you need to be aware of the limitations and challenges of your tissue/specimen type. Also, it is important to gauge the amount of initial RNA content in a specific cell type when preparing cell suspension for single-cell RNA sequencing, as this affects the number of cells that will be used for further processing. The cells in the suspension need to be counted using a hemocytometer or an automated cell counter. Also, the number of dead cells has to be estimated and removed from the cell suspension to ensure a good number of recovered cells. Further, the cells should be handled carefully and at optimal temperatures. For example, pipetting the cells through a small pipette tip can cause physical damage to the cells. Sponsored by: References 1. Levy SE, Myers RM. Advancements in next-generation sequencing. Annu Rev Genomics Hum Genet. 2016;17(1):95-115. doi: 10.1146/annurev-genom-083115-022413 2. Chin ELH, da Silva C, Hegde M. Assessment of clinical analytical sensitivity and specificity of next-generation sequencing for detection of simple and complex mutations. BMC Genet. 2013;14:6. doi: 10.1186/1471-2156-14-6 3. Ascierto PA, Bifulco C, Palmieri G, Peters S, Sidiropoulos N. Preanalytic variables and tissue stewardship for reliable next-generation sequencing (NGS) clinical analysis. J Mol Diagnostics. 2019;21(5):756-767. doi: 10.1016/j.jmoldx.2019.05.004 4. Auer H, Mobley JA, Ayers LW, et al. The effects of frozen tissue storage conditions on the integrity of RNA and protein. Biotech Histochem. 2014;89(7):518-528. doi: 10.3109/10520295.2014.904927 5. Scrivano S, Sanavio M, Tozzo P, Caenazzo L. Analysis of RNA in the estimation of post-mortem interval: a review of current evidence. Int J Legal Med. 2019;133(6):1629-1640. doi: 10.1007/s00414-019-02125-x