Jeffrey Perkel has been a scientific writer and editor since 2000. He holds a PhD in Cell and Molecular Biology from the University of Pennsylvania, and did postdoctoral work at the University of Pennsylvania and at Harvard Medical School.
Unless you do your lab work under a rock – heck, maybe even if you do -- you’re well aware of the benefits of so-called “next-generation sequencing” (NGS) technologies. Whether it’s production-scale equipment like Illumina’s HiSeq®, or more personalized instruments like Life Technologies’ Ion Torrent PGM, these NGS instruments are unscrambling genomes and transcriptomes at a rate that was literally unimaginable a decade ago.
Most of the attention, of course, has focused on the instruments themselves and what they can do. But to reap those benefits, researchers first need to have a sequence-ready sample, and that involves more than mere DNA isolation.
Enter NGS sample preparation tools. Available from both sequencer manufacturers and third-party vendors, these reagent kits simplify and standardize the process of converting a DNA sample into a sequencing library, and, if desired, preparing it for multiplexing. Also available are target selection systems, for those interested in sequencing only exomes or methylated regions, for instance.
Naturally, given the incredible growth in this application space, there’s no shortage of tools available. Whether you prefer to do your work manually or using automated liquid handlers, you’re sure to find one that fit your needs.
Key steps in NGS library preparation
A sequencing library is simply a collection of DNA fragments that are the correct length and contain the appropriate adaptor sequences to be processed by a sequencer. The precise steps required to make those libraries, of course, depends on both the application and the instrument. Transcriptome libraries, for instance, require cDNA synthesis, whereas chromatin immunoprecipitation (ChIP)-seq libraries require, well, ChIP. Likewise, it takes different steps to produce a paired-end library compared to one that will be read in one direction only.
But in general, says Mark Gardner, Vice President and General Manager of Advanced Genomic Systems at Life Technologies, the process usually includes a set of core steps regardless of platform: DNA extraction, fragmentation, and size-selection; fragment end processing and clean-up; ligating adaptor sequences; and amplifying the resulting pieces.
The DNA fragmentation step typically is accomplished using sonication, though enzymatic alternatives (such as New England Biolabs’ NEBNext® dsDNA Fragmentase®), are also available. Life Technologies recommends its Ion Torrent customers use the Diagenode Bioruptor® UCD-200 sonicator to fragment their samples, whereas SOLiD customers are directed to use the Covaris™ S220 sonication system instead. Illumina also recommends Covaris sonicators for users of its TruSeq sample preparation kits, as well as systems based on nebulization.
For size selection, Gardner recommends Life Technologies’ E-Gel® SizeSelect™ Gels or Pippin Prep™ kits, both of which use agarose gels. Illumina suggests Beckman Coulter AMPure XP beads for the same purpose.
Once you have your size-selected, fragmented DNA, the next step is the library preparation itself. Illumina offers several options under its TruSeq brand name, including the TruSeq DNA Sample Preparation Kit and TruSeq RNA Sample Preparation Kits v2. In the TruSeq workflow, the fragment “clean-up” step involves first blunt-ending the fragments’ ragged ends and then A-tailing, in which the fragments are tagged with 3’-adenine residues to enable ligation with 3’-T-containing adaptors.
These adaptors contain the barcodes, or index sequences, that Illumina uses for multiplexing reactions, says Jeremy Preston, Illumina’s Director of Product Marketing for sequencing systems and consumables. The standard “low-throughput” TruSeq DNA LT kit includes 24 index sequences, whereas the newly released TruSeq DNA HT kit has 96.
“You prepare each sample in a different well, and then you can mix them all together and run them in one lane,” explains Preston. “There are no extra steps in the actual assay, because when you add the adaptor, you include the barcode.”
Illumina also has another line of kits under the Nextera brand name. Acquired in the company’s purchase of Epicentre Biotechnologies, Nextera is a rapid “transposome”-based technology that fragments and tags DNA simultaneously, a process the company calls “tagmentation.” That first step is accomplished in “as little as 15 minutes,” according to company literature; a second PCR step adds sequencing primers and up to 96 barcodes. Thus, the whole protocol is far faster than the multi-step TruSeq: Whereas the TruSeq workflow typically takes 8 to 12 hours, Nextera can be completed in just 90 minutes. It uses far less input DNA, too, 50 ng instead of 1 µg.
Life Technologies likewise offers a portfolio of sample preparation and barcoding kits including the Ion Xpress™ Plus Fragment Library Kit and NuGEN® Encore™ Library System for Ion Torrent, as well as the SOLiD® Fragment Library Construction Kit. Roche Applied Sciences’ collection includes the GS FLX Titanium Rapid Library Preparation Kit and related reagents.
Researchers can also acquire reagents from third-party vendors. New England Biolabs offers more than 35 DNA and RNA library preparation NEBNext kits for Illumina, Ion Torrent, SOLiD, and Roche/454, says New England Biolabs Product Manager Fiona Stewart. “We are platform-agnostic,” she says. These kits differ in the enzymes, adaptors, and protocols they contain, whether they use blunt-end ligation or A-tailing, and so on.
The kits also differ in how they are formulated, says Stewart. Though users can opt to purchase full sets of preformulated master-mixes, the company also offers unmixed reagent sets and modules for individual steps of the workflow, as well a custom and bulk options, to accommodate researchers who wish to modify workflows for their specific samples. “We can formulate the reagents any way they prefer,” she says.
Reduce labor and increase reproduciblity with automation
Even with kits, NGS sample prep is a complicated, multi-step, and laborious process, says Alisa Jackson, Marketing Manager for genomic solutions at Beckman Coulter Life Sciences. Thus, it’s easy to make mistakes and/or introduce variability, especially when handling multiple samples.
“Next-gen sequencing has definitely brought down the cost of DNA analysis and increased throughput, but there is still complexity in the sample prep,” says Jackson. “And it is expensive enough that people don’t want to waste time and money doing it manually.”
Some sequencer companies, like Illumina, produce kits in automation-ready formats. And automation vendors like Beckman Coulter have developed systems to use them. Beckman also offers reagents for library construction, and so provides complete automated solutions for low to high- throughput processing. Four validated solutions are offered for this purpose: the low-throughput SPRIworks Fragment Library Systems I, II, and III (for Illumina platforms, Roche 454, and SOLiD 4400 and Ion Torrent 100 base pair reads, respectively), and the higher throughput SPRIworks HT (which is customizable, but configured for Illumina by default).
SPRIworks Fragment Library Systems I–III, which are run on a single SPRI-TE liquid handling instrument, are closed solutions. They include methods and reagents loaded in a cartridge that are specific for each NGS platform, explains Jackson. The system can process 10 samples simultaneously through the four key steps of library construction, she says – end-repair, A-tailing, ligation, and SPRI (Solid Phase Reverse Immobilization)-based size-selection, as well as clean-up steps – in about 4-5 hours.
The SPRIworks HT, on the other hand, is essentially a pre-configured, turnkey Biomek FX system with both Span-8 and Multichannel-96 pipetting heads. The solution includes a reagent kit for library construction and PCR setup and cleanup. The SPRIworks HT performs the same steps as the SPRIworks I–III, as well as PCR setup for the final amplification step. It also provides a suite of methods for qPCR setup, normalization and pooling for multiplexed sequencing. It can generate 96 library samples in 6 hours with size selection, or 4 hours without size selection, Jackson says. By default, the SPRIworks HT is configured to handle Illumina library prep, but because it is based on the open Biomek platform, the system can be programmed and customized to accommodate other protocols as well.
According to Jackson, the SPRIworks HT system’s on-board, SPRI–based size-selection capability is a significant, and configurable, distinguishing factor. “We can do 96 samples and size-select in any of four ranges: none, small, medium, and large. And you can specify that well-by-well.”
Life Technologies also offers kits prepackaged for automated liquid handlers. Or, for a “soup-to-nuts” alternative, Gardner recommends the Applied Biosystems Library Builder™. The Library Builder performs all the steps of library preparation, but according to product literature, it also can perform upstream DNA purification steps, “reducing hands-on time by 50%.”
Focus on your region-of-interest with target enrichment
Falling prices and rising data output notwithstanding, the fact remains that NGS experiments remain expensive. Many users thus aim to squeeze as much sequence on as many samples as possible out of each run. One solution, of course, is barcoding: by adding unique sample tags or index sequences to each fragment, users can mix samples and let the bioinformatics software figure it all out on the back-end.
Another option, often paired with barcoding, is target enrichment, for instance to sequence only specific genes or even the entire exome. That way, users need less sequence per sample and thus can handle more samples per run.
One popular option is Agilent Technologies’ SureSelect™ system, in which biotinylated RNA oligonucleotides capture specific regions of interest. The resulting RNA:DNA hybrids are then captured on streptavidin beads.
Agilent offers off-the-shelf products for the 30 Mbp human exome (SureSelectXT Human All Exon v4) and the X chromosome, but custom designs are also available, says Olle Ericsson, the company’s Marketing Director for NGS products. So, too, is a reagent set for sequencing methylated regions (SureSelectXT Human Methyl-Seq).
Agilent also manufactures a custom multiplex PCR-based enrichment technology, called HaloPlex, which can enrich up to 5-Mbp of sequence. HaloPlex is used in clinical research applications where simplicity and reproducibility are key, Ericsson says, in particular in combination with benchtop sequencers. “SureSelect offers a higher degree of customization including alternative organisms and RNA analysis for research applications,” he says.
Life Technologies and Illumina also offer solution hybridization-based targeting products, through their TargetSeq™ and TruSeq brands, respectively. The latter can collect between 0.5 and 25 Mbp per sample. And, like Agilent, LifeTech offers a PCR-based alternative called AmpliSeq (available for cancer and inherited diseases, as well as in custom designs).
Other target-enrichment solutions are also on the market, or soon will be. Qiagen, for instance, will shortly launch reagents to remove ribosomal RNA from total RNA samples to improve the sensitivity of RNA-seq applications, says Dirk Loeffert, Qiagen’s Vice President and head of sample and assay products. According to Loeffert, the GeneRead rRNA Depletion Kit is based not on biotinylated hybridization probes as other systems are, but rather an RNA:DNA hybrid-specific antibody. That, he says, ensures highly specific pull-down only of the ribosomal sequences in the mix. “We get greater than 99.9% removal of rRNA,” he says.
Qiagen’s soon-to-be-released GeneRead DNAseq NGS Panel System will also address target enrichment, using multiplex PCR to pull out tens to hundreds of gene-coding sequences at once. The system will include an “automated data analysis tool,” says Loeffert.
Whatever your sequencing platform or application, users should not be intimidated by NGS library preparation, says Preston. The technology has had several years to mature, and the protocols well tested. But, as with any new procedure, he does recommend customers study the protocols thoroughly before diving in the first time, as they are extensively vetted.
“There are some points where you have to follow the protocol carefully and not diverge from it,” he says. Whatever your application, that’s good advice.
The image at the top of this page is of Beckman Coulter's SPRIworks HT.