This is a myBaits enrichment kit for performing hybridization capture of a target of interest prior to sequencing. Briefly, total sample RNA must first be converted to a fully sequenceable, barcoded NGS library via any appropriate commercially-available NGS library prep kit (typically, RNA > cDNA > library). Then the barcoded NGS library (or pool of libraries) is enriched following the myBaits reagents and recommended protocol. Then a universal library amplification is performed, and the library (or pooled libraries) can be taken to sequencing on any NGS platform (typically Illumina, but long-read sequencing platforms are also supported). Continue reading below to learn more about the hybridization capture technique and its many advantages!
What is Hybridization Capture with Custom Probes?
Next-generation sequencing (NGS) is a powerful method which facilitates rapid deep sequencing of DNA or RNA samples. However, within a whole genome or metagenome, the actual sequence regions relevant for a given research project are typically of low relative abundance, which would necessitate deep shotgun sequencing to accurately detect and reconstruct them. Therefore a targeted approach NGS to increase read coverage just in genomic regions of interest is highly desirable to improve cost-effectiveness and allow for many more samples to be assayed. There are many NGS techniques that help to reduce per-sample sequencing costs (such as single- or multi-plex PCR amplicons, GBS/RAD-seq, etc), but only hybridization capture of NGS library molecules using biotinylated probes provides the best overall balance of ease, efficiency, and cost-effectiveness. By designing a myBaits Custom kit for your next targeted sequencing project, you can bring down the cost per sample AND increase the accuracy and power of your data analysis. Highly effective myBaits hyb capture probes can be designed to enrich a target as small single locus for a single organism, or as broad as tens of thousands of loci from an entire taxonomic group.
Figure 1. How does myBaits work?
(1) HYBRIDIZATION – An NGS library is denatured via heat, and allowed to hybridize to a complex mixture of complementary biotinylated RNA baits over the course of several hours. Adapter-specific blocking oligos prevent random annealing of library molecules at the common adapter sites.
(2) WASHING – After the hybridization is complete, the biotin present on each bait is bound to a streptavidin-coated magnetic bead. Wash steps help remove off-target or poorly-hybridized library molecules.
(3) AMPLIFICATION – The remaining library molecules that are still bound to their complementary baits are denatured via heat, and amplified using universal library primers. This “enriched” library can now be sequenced.
Advantages of Hyb Cap Technology
There are many advantages to the hybridization capture approach, which have made it a workhouse technique for both routine and complex workflows for modern NGS research. While hybridization capture reagents are available from multiple vendors, only myBaits® Custom kits from Arbor Biosciences offer the optimal balance of flexibility and performance. Our proprietary oligo synthesis platform allows us to manufacture fully customized probes at any scale and level of complexity, making them suitable for any project type and budget. We are experts in the design of hyb capture probes, and our team of scientist project advisers have years of experience with an enormous variety of sample types and project goals, such as ancient DNA, metagenomics, microbiology, phylogenetics, environmental DNA, pathogen sequencing, RNA-seq, and more. And if our free probe design services and easy-to-use kits are not enough, you can even outsource your entire enrichment project to our in-house team to handle for you. Whatever the targeted sequencing need, Arbor Biosciences has the right solution for you.
- Straightforward procedure fits in between NGS library prep and sequencing
- Simple to design custom probes from one or more reference sequences
- Probe sequences do not have to be exact match to targets (>20+% divergence can be tolerated)
- myBaits hyb cap kits contain all reagents necessary to perform protocol
- No special equipment or training necessary to perform
- Can use with any NGS platform, including both short- and long-read sequencing
Figure 2. Hybridization capture tolerates both rearrangements and sequence variation.
Both hyb cap and amplicon sequencing can effectively enrich for known sequence regions. However, only hyb cap can retrieve target sequences that have significant rearrangements and/or mutations relative to the reference used for probe design, for example when capturing genomic content from novel microbial genomes.
All myBaits kits are for research-use only, and are not validated for diagnostic or therapeutic purposes.