Next-generation sequencing (NGS), which accomplishes high-throughput DNA sequencing using massive parallelization, requires input of a pure starting sample of DNA for output of better results. This is crucial for NGS sample-preparation steps, such as target enrichment and NGS library generation. Impurities or contamination in the starting material can lead to artifacts or even the need to redo the entire NGS run, wasting time and money.

Although DNA purification has become routine for many applications, the preparation of genomic-DNA samples for NGS is not a step to take shortcuts. “When it comes to your downstream NGS applications, it’s “garbage in, garbage out,” says Curtis Knox, global strategic marketing manager for NGS at Promega. “Navigating through limited, precious samples, sensitive assays and time constraints requires great sample quality going into your downstream sequencing—in short, every step counts in the overall NGS workflow.”

Here we discuss some new tools for preparing genomic DNA for NGS and some tips that may help along the way.

Sample types

It is important to select a sample-preparation tool that is designed for the type of sample that is being analyzed. Every sample type has unique characteristics, which can also present unique challenges, and DNA-extraction kits are typically geared toward solving these. Kits for extracting genomic DNA from all types of samples abound; the following are some of the newer ones.

FFPE samples

A common sample type, formalin-fixed paraffin-embedded (FFPE) tissue, definitely requires its own preparation tools to address its particular challenges, such as “DNA fragmentation, formalin-induced cross-linking and base deamination,” says Markus Sprenger-Haussels, senior director, head of sample technologies life sciences at QIAGEN. QIAGEN’s new GeneRead DNA FFPE Kit removes cytodine deamination artifacts—particularly important for NGS, as deaminated cytosine will be falsely read as thymidine during sequencing—that can occur in FFPE samples as a result of fixation. This helps “avoid false mutation-detection reads when analyzing the sequence,” says Sprenger-Haussels.

Other tools for extracting DNA from FFPE samples include Covaris’ truXTRAC™ FFPE DNA Kit, MO BIO Laboratories’ BiOstic® FFPE Tissue DNA Isolation Kit, Norgen Biotek’s FFPE DNA Purification Kit, Thermo Fisher Scientific’s MagMAX™ FFPE DNA Isolation Kit and Zymo Research’s ZR FFPE DNA MiniPrep™.

Small samples

Extremely small samples pose the challenge of extracting enough DNA while maintaining high enough quality for NGS. Thermo Fisher Scientific’s new Picopure DNA kit is designed to extract DNA from extremely small starting samples—as few as 10 cells—derived from cells in culture, sorted cells, FFPE tissue, embryos or cells from laser capture microdissection (LCM), according to Vidya Venkatesh, product manager in genetic sciences at Thermo Fisher Scientific. Rather than lose sample during a purification step, the kit instead uses an optimized extraction process so that no subsequent purification step is required. “This maximizes your yield and streamlines the workflow, as you can take your lysate directly into our Ion Ampliseq library preparation,” says Venkatesh.

With plasma and other types of noninvasive sample types becoming more prevalent, Promega’s new Maxwell® RSC ccfDNA Plasma kit is designed to purify circulating cell-free DNA (ccfDNA). Also available in a high-throughput version, the kit uses a new chemistry that targets small-sized ccfDNA and minimizes contamination with genomic DNA. For extracting and purifying genomic DNA for NGS, the company offers a range of purification tools, including membrane-based columns in its Wizard® and ReliaPrep™ Purification kits and magnetic resin-based chemistries in its Maxwell® systems. Another tool for ccfDNA purification is Norgen Biotek’s Plasma/Serum Cell-Free Circulating DNA Purification kit.

Microbiome samples

QIAGEN’s new QIAamp DNA Microbiome Kit is designed for microbiome researchers who use samples collected from swabs and bodily fluids. “Microbiome analysis is often impaired by contamination of host DNA, which can make up to 90% of all sequencing reads in a swab sample,” says Sprenger-Haussels. QIAGEN’s kit addresses this by removing contaminating host DNA using two different host-DNA removal steps. Another microbiome tool is New England BioLabs’ NEBNext® Microbiome DNA Enrichment Kit.

In addition, MO BIO Laboratories has a series of kits also designed for microbiome researchers that are optimized for optimal extraction and preservation of samples and the assays are scalable and ammeble to automation platforms. These kits have been used in promenient microbiome studies including the Human Microbiome Project and Earth Microbiome Project.

Other factors to consider

In addition to sample type, another factor to consider before choosing a preparation method is the type of NGS application in which the DNA sample will be used. NGS applications such as whole-genome or exome sequencing are compatible with most DNA-purification kits (assuming appropriateness for sample type). Other NGS applications, such as de novo sequencing and genome-finishing applications, produce longer reads and may benefit from longer DNA templates. Tools like QIAGEN’s new MagAttract HMW DNA Kit, which purifies highly intact DNA molecules from tissue, cells or blood, preserve the integrity of long (100- to 200-nucleotide) DNA fragments using magnetic beads and mild lysis conditions.

Recent advances in NGS technology, however, are making DNA fragment length less of a concern for NGS applications. For example, Illumina’s TruSeq Synthetic Long-Read technology on its HiSeq platform constructs synthetic long reads from shorter sequencing reads. So-called third-generation sequencing technology, such as PacBio’s single-molecule real-time sequencing (SMRT), also generates long sequencing reads. Thus, if long reads are important in your research, improvements in both sample preparation and NGS technology are available.

Future directions for NGS sample preparation

The future of preparing DNA samples for NGS will be influenced by two driving needs of researchers today. One is the need to obtain quality DNA from smaller and smaller sample sizes. “As NGS becomes ever more sensitive, DNA-isolation methods will also get more efficient in handling small sample sizes,” says Sprenger-Haussels. The other need is to keep the “doing” of NGS sample preparation simple. “With NGS becoming more widely available and less costly, and researchers wanting to start with less and less material, we see the need to have a gDNA method that is sensitive enough to be incorporated into a simplified workflow,” says Venkatesh.

Increasingly, researchers are recognizing that a population of cells of the same cell type is not necessarily homogeneous—such as in tumors, for example. NGS of small samples of a cell population, or even of single cells can reveal heterogeneity in genomic DNA. Knox believes that such applications will grow rapidly in the next few years. “Researchers are pushing the limits of what they can detect while recognizing that samples are not necessarily homogeneous,” he says. “It’s important to be able to see what’s happening in a very small sample.” NGS is skyrocketing in the translational and clinical research fields, according to Knox, and with no signs of stopping. Thus it’s more important that researchers select their NGS preparation tools carefully—by considering their sample types and their particular NGS application.