A Scientific Update

Fall/Winter 2017 expressions a scientific update ® ! No witchcraft required in this issue – Clone with Confidence 2 Overcoming the challenges of applying target enrichment for translational research 5 Now available: the NEBNext Direct® BRCA1/BRCA2 Panel 6 NEBNext® Ultra™ II RNA Library Prep kits – Streamlined workflows for lower input amounts 8 No witchcraft required: Special Prices on all Restriction Enzymes and more! 14 Get free tube opener with NEB competent cells 16 New to Molecular Biology? Get your free NEB Starter Pack be INSPIRED drive DISCOVERY stay GENUINE FEATURE ARTICLE Overcoming the challenges of applying target enrichment for translational research by Andrew Barry, M.S., New England Biolabs, Inc. Target enrichment is used to describe a variety of strategies to selectively isolate specific genomic regions of interest for sequencing analysis. The wide array of approaches presents challenges in selecting the appropriate technology for the growing number of research and clinical applications to which the sequencing data will ultimately be applied. INTRODUCTION strates the practical need for continued use of Examples of these hybrid approaches include In recent years, several techniques have emerged target enrichment strategies across the gamut of multiplex extension ligation (3), molecular to enrich for specific genes of interest. When translational research activities. inversion probes (MIPS)/padlock probes (4), determining the appropriate target enrichment nested patch PCR (5), and selector probes (6). technology to use, one must first consider the TARGET ENRICHMENT These technologies can be broadly characterized primary goal of the study. For example, different APPROACHES as having more complex workflows, requiring approaches will be used if the aim is to identify splitting of samples into separate reactions, and known variants already shown to have clinical There are a number of different target enrich- creating challenges in target coverage uniformity. implications, versus discovering novel nucleic ment approaches that can be grouped into three generalized categories: in-solution hybridization, acid variants that may be associated with a given NEBNext Direct® for target enrichment phenotype. Variant identification lends itself multiplex PCR, and “alternative approaches”, which span a wide variety of techniques. NEBNext Direct is a novel target enrichment to more focused enrichment strategies, while method that addresses several drawbacks that variant discovery is driven by trade-offs between In-solution hybridization-based approaches, exist in alternative enrichment technologies sequencing costs and target territory, as well as originally developed for whole exome sequenc- (Table 1). Enrichment is achieved through available sample cohort sizes for a given study. ing, use biotinylated oligonucleotides to capture direct hybridization of biotinylated DNA baits As translational research seeks to bridge funda- genomic regions of interest (1). Commercial- to denatured, fragmented molecules, which mental laboratory research and clinical treatment ly-available kits use DNA or RNA baits ranging are subsequently captured using magnetic regimens for patients, there is an emerging from 50-150 nucleotides. Researchers have streptavidin beads (Figure 1, page 3). Unlike need to balance discovery of novel nucleic acid adapted this technique for more focused panels, alternative in-solution hybridization protocols, variants, identification of known variants, and ranging down to tens of kilobases in target the NEBNext Direct protocol does not require studies aimed at revealing associations with clin- territory, with limited success in maintaining library preparation prior to hybridization of ical phenotypes. Recent advances in sequencing specificity for target regions. oligonucleotide probes. This feature reduc- technologies have revolutionized the field of ge- Multiplex PCR-based enrichment is most often es the overall amount of amplification that is nomic research, making tractable the application employed for highly focused panels targeting a required throughout the protocol and enables of whole genome and whole exome sequenc- smaller territory than in-solution hybridization, single-stranded DNA to be captured along with ing for broad discovery of germline genomic and is typically limited to 150-200 amplicons denatured, double-stranded DNA. variants. However, despite these advances, the (2). Using a pool of primers, enrichment is Conversion of captured fragments to sequence- oncology field is fraught with the complexity of accomplished through PCR amplification of the ready libraries is achieved by the ligation of detangling the underpinnings of tumorigenesis, targeted regions, which is followed by adaptor a loop adaptor to the proximal 3´ end of the progression, and resistance mechanisms driven ligation or a second round of PCR using tailed captured molecule. During these steps, the bait / by somatic variants present at extremely low primers to include sequencing adaptors. Scaling target molecules remain bound to the magnetic abundance in mixtures of malignant and stromal this technology has presented a challenge in streptavidin beads and are processed in a single cells. These complexities necessitate increases in maintaining target coverage uniformity. reaction tube. This eliminates sample loss and the depth of sequencing coverage to confident- A number of alternative approaches have been improves overall conversion efficiency. ly call somatic variants, making broader scale developed in an attempt to bridge the gap be- approaches infeasible from an economic and tween hybridization and PCR-based approaches. practical standpoint. To overcome these challenges, focused gene panels are being applied to patient samples. The size TABLE 1: of the panel is highly variable, trending toward Enrichment Challenges and Advantages of NEBNext Direct decreased genomic content as assays progress from pure research and discovery applications to Challenge NEBNext Direct Advantage clinical diagnostic assays. Furthermore, clinical Specificity across panel sizes Enzymatic removal of off-target sequence applications raise the question of incidental findings and how to report them, introduc- Uniformity of coverage Individual synthesis of baits & empirical balancing ing challenges for diagnostic assays based on Sensitivity to detect variants Unique Molecule Indexes for PCR duplicate marking & consensus variant calling sequencing entire genomes. This trend demon- Degraded or low quality samples Short baits that extend across molecules, targeting both DNA strands 2 Following ligation of the 3´ adaptor, the bait FIGURE 1: is extended across the entirety of the captured NEBNext Direct target enrichment workflow molecule, resulting in double stranded DNA that is ready for ligation of the 5´ unique molecular 5´ 3´ Genomic identifier (UMI) adaptor. This adaptor contains 3´ 5´ DNA a 12 bp random sequence that is incorporated Target region discretely into each molecule, indexing each Fragmentation molecule prior to amplification. This index can 5´ 3´ be used to identify duplicate molecules, thereby 3´ 5´ reducing artifacts that can lead to false positive variant calls. Denaturation 5´ 3´ probe hybridization Biotin probe targets both 3´ 5´ Once the 5´ adaptor is ligated, the 3´ loop strands (shown for 1 strand) adaptor is cleaved, and the target molecule is PCR Biotin probe amplified off of the bait complex. It is important to note that the bait strand is not perpetuated 3 blunting of DNA 5´ 3´ Enzymatic removal through the PCR amplification and is not present 3´ 5´ of off-target sequence in the final, sequencer-ready library. Streptavidin bead The coverage plots of NEBNext Direct libraries are unique for a hybridization-based approach dA-tailing 5´ A 3´ in that reads have a defined 3´ end, resulting in 3´ 5´ coverage plots that resemble PCR-based libraries, yet the approach allows for flexibility in tiling across longer targets. Disambiguation of PCR duplicates is accomplished by two features of the Ligation of 3 adaptor 5´ A NEBNext Direct library: A variable 5´ end and a 3´ T 3´ adaptor 12 bp randomized UMI that is incorporated into the 5´ adaptor. 5 blunting Probes are extended to 5´ A CHALLENGES OF TARGET of DNA the 5 end of the randomly 3´ T sheared fragment, creating ENRICHMENT FOR a variable 5 end of the TRANSLATIONAL RESEARCH target read Specificity of target enrichment Ligation of 5 Unique Molecular 5´ A For any study that necessitates enrichment of spe- 5 UMI adaptor Identifier (UMI) addition 3´ T cific targets over more comprehensive sequencing 5´ UMI improves sensitivity approaches, specificity becomes more important as adaptor it directly translates to the amount of sequencing required to achieve the minimum coverage thresh- Adaptor cleaving 5´ A 3´ old to reliably detect variants of a given frequen- 3´ T 5´ cy. Specificity is typically measured by looking at the percentage of sequencing data that is derived from the targeted regions relative to the data that PCR amplification 5´ A 3´ is aligned to other parts of the reference genome. 3´ T 5´ Enrichment of genomic regions is typically Sequencing-ready fragment Incorporated sample index achieved by either amplifying the desired regions through PCR to generate enough copies of the targeted regions over the untargeted regions, in which randomly fragmented molecules are Uniformity of coverage across targets or through hybridization of complementary captured overnight, and without any removal

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