Caitlin Smith has a B.A. in biology from Reed College, a Ph.D. in neuroscience from Yale University, and completed postdoctoral work at the Vollum Institute.
Next-generation sequencing (NGS) has turbocharged genetics. Its unmatched throughput levels increasingly are driving a variety of applications. To reap the benefits of NGS, you need to prepare a sample that is compatible with your particular sequencing platform. Luckily, it has never been easier to turn your raw DNA sample into a proper NGS-compatible library.
Library-preparation kits
Next-gen sequencers work with DNA samples in “library” format. DNA libraries are created by extracting the sample DNA and cutting it into fragments by either sonication or enzymes. DNA fragments that are the right size for your sequencer are then selected, either by agarose gels or magnetic beads. Next, the DNA fragment ends are trimmed, and adaptors are placed at 3’ ends. The adaptors make the library compatible with the sequencer and also serve as indices, or tiny “barcodes,” for identification in multiplexed experiments—as many as 384-plex from Illumina, for example. Today, vendors offer improved library-preparation kits to make the job easier.
Though New England Biolabs (NEB) does not manufacture next-gen sequencers, it does offer a wide range of sample-prep tools, including the NEBNext® kits (DNA or RNA) for Illumina, Life Technologies and Roche/454 sequencers. According to Fiona Stewart, product marketing manager for next-generation sequencing at NEB, the NEBNext Ultra™ DNA- and RNA-prep kits are well suited for prepping libraries from a broad range of sample types, including potentially compromised samples, such as formalin-fixed, paraffin-embedded (FFPE) tissue, or from very low input amounts. “As more challenging FFPE samples are being sequenced, investigators are highlighting the need for robust protocols for FFPE and other difficult samples that can reliably generate high-quality libraries despite low-quality nucleic acid inputs,” says Stewart.
Illumina offers two workflow options for NGS sample-prep kits. The TruSeq line of kits is better suited for whole-genome sequencing with high quality and coverage, to “arrive at a set of the most accurately called and reproducible variants,” says Daniel Peiffer, senior market manager for DNA applications at Illumina. The TruSeq kits include sample indices for multiplexing samples as well as beads for selecting DNA-fragment sizes. New additions to this line include the TruSeq DNA PCR-Free kit and the TruSeq Nano DNA kit.
In contrast to TruSeq, which takes five to six hours to generate libraries, Illumina’s Nextera kits are made for fast, easy workflows that can produce complete libraries in less than 90 minutes. The Nextera technology derives speed from multitasking—it accomplishes DNA fragmentation and addition of adaptors simultaneously via a transposase-based step the company calls “tagmentation.” Nextera tools are best for work that needs to be fast and with minimal hassle, such as if “you have a pool of homemade amplicons that you would like to sequence across hundreds of samples ... on a MiSeq [sequencer], and completing this project as quickly as possible is paramount,” says Peiffer.
Illumina also offers TruSeq tools for working with RNA. Though whole-transcriptome options are available, the new TruSeq Targeted RNA kit enables targeted gene-expression profiling of 12 to 2,000 targets in one experiment.
More specific targets
Target enrichment lets researchers zero in on genome (or transcriptome) regions of interest, which increases sensitivity in detecting variants as well as throughput. “The more uniform the sequence coverage provided by an enrichment technology, the more samples that can be multiplexed in one sequencing run while retaining the same sensitivity for variant detection,” says Ji Wu, international marketing director of Roche Sequencing Solutions at Roche NimbleGen.
Target enrichment is performed today using two primary methods, depending on your experiment type. The first is amplicon- or PCR-based enrichment, embodied in Agilent Technologies’ HaloPlex, Life Technologies’ AmpliSeq™ and QIAGEN’s GeneRead DNAseq Gene Panels. This method is generally faster and easier than hybridization-based alternatives and so more suited for quick gene panel screens, for example. “Amplicon-based tools are good for finding simple variants when you have a precise idea of what kind of variants you are looking for, and where these variants are likely to be found,” says Wu. On the other hand, this method may miss indels and translocations, and is prone to bias or amplification artifacts.
Hybridization-based target enrichment, often used in whole-exome sequencing, is better at detecting a wide range of variants with high fidelity, such as copy-number variants, indels, translocations and SNPs in or near the target regions. “For certain regions of the genome or certain types of sequences, it can be quite challenging to design unique and effective PCR primers, but hybridization-based approaches tend to be much less sensitive to such constraints,” says Wu.
Roche NimbleGen’s hybridization-based tools “incorporate a minimum of 2.1 million probes for standard designs, which provide superior flexibility for probe placement that is the key to achieving uniform sequence coverage and more effective SNP calling,” says Wu.
In addition to Roche, hybridization-based target-enrichment tools include Agilent’s SureSelect, Life Technologies’ TargetSeq™ and Illumina’s TruSeq and new Nextera Rapid Capture Enrichment kits.
One application now benefitting from target enrichment is microbiome analysis, which previously was often hampered by contamination with vastly more abundant host DNA. “This host-DNA contamination problem made it prohibitively expensive to sequence the microbiome of many areas of the human body, including saliva and mid-vagina samples,” says Stewart. “The NEBNext Microbiome DNA Enrichment kit enables sequencing of these previously elusive microbiome samples, and we and our customers have applied this host methylation-based enrichment method to a number of sample types in humans and other organisms.”
Automation for sample prep
Automating NGS sample prep makes life easier for researchers while dramatically reducing the chance of human error and increasing throughput. Automation combined with sample-prep kits can speed DNA purification, library construction and fragment-size selection. It can also facilitate trickier protocols, such as extracting nucleic acids from FFPE samples.
Beckman Coulter’s SPRIselect, a magnetic bead-based technology for gel-free size selection of DNA fragments, is now automated on the company’s standard Biomek 4000 and on higher-throughput Biomek FXP platforms. Alisa Jackson, global marketing manager for sample preparation at Beckman Coulter Life Sciences, says SPRIselect “is a flexible kit that enables researchers to select a wide range of fragment sizes by executing left, right or double size-selection cutoff points.”
Beckman Coulter has also partnered with Illumina to provide automation for that company’s NGS sample-prep kits, including the TruSeq, Nextera and Epicentre lines. The most recent release is the TruSeq Stranded mRNA kit.
Jackson says sample-prep kits have become simpler and more robust, which makes them more amenable to automation. Illumina’s PCR-free kit for DNA and Nextera kit, for instance, “are formatted for high-throughput processing and are therefore easier to automate, increasing efficiency and data confidence in the labs.”
Beckman Coulter also is developing automated systems for small and medium labs. “Biomek 4000 methods for qPCR setup and AMPure XP cleanup provide researchers with a low-cost solution for automating critical steps in the NGS sample-prep process,” says Jackson. “Each method can process up to 192 high-quality samples, in less than two hours, that are ready to move on to downstream normalization, pooling and enrichment steps.”
Rather than automate the entire process, Advanced Analytical Technologies, Inc., has developed an instrument for automating sample-quality assessment. The Fragment Analyzer™ Automated CE System uses capillary electrophoresis and intercalating fluorescent dyes to quantify and determine sizes of the nucleic acids in a sample. The system can run 12 or 96 samples simultaneously and resolve DNA fragments of 10 bp to 40,000 bp with up to 2-bp resolution.
Whether automated or not, NGS is becoming increasingly common, and sample-prep tools are sure to become even more important. “As NGS continues to be adopted more widely as a standard technique, the amount of multiplexing, and therefore the number of libraries being made, is also increasing,” says Stewart. “Advances in speed of library prep, combined with the development of more robust methods and cost reductions, [are] enabling more sequencing labs to increase their throughput.” Stay tuned for even more NGS tools to emerge in this quickly evolving field.