Friday, 13 March 2015

A better way to sequence exomes?

I caught up with a new company on the target capture scene, Directed Genomics, at AGBT. Their approach is based on a simple idea: if you want to sequence exomes, why not capture only exons?

Most exome-seq methods (Illumina, Agilent, Nimblegen) use oligo-baits to pull-down adapter-ligated fragment libraries, with fragments of 200-300bp. As exons are only 170bp long (80–85% Human exons less than 200bp Zhu et al & Sakharkar et al) we sequence lots of near- or off-target bases. These can be used (cnvOffSeq for instance), but are to some degree wasted sequencing.

The Directed Genomics approach: similar to other exome capture companies Directed Genomics also uses a probe hybridisation to targeted regions and/or exons, but applies this in a very different manner than we’re used to with standard exome capture. Two methods are presented in their recent posters; the first uses two probes, one at each end of the exon; the second uses a single probe hyb and random 5’end to create molecularly identifiable libraries. Current plans appear to be for custom panels, but hopefully they'll to build out to a whole exome panel over time.

Directed Genomics workflows

1: In their dual-probe method a short 50bp biotinyated-oligo probe is hybridised to fragmented gDNA at the 3’ end of an exon, the sequence upstream of this is then enzymatically digested and the 3’ hairpin adapter ligated. Next a second 50bp probe is hybridised to the 5’ end of the exon, the 5’ end is blunted and a 5’ adapter is ligated. Rather cleverly the hairpin adaptor ligated at the 3' end of the target links the target to the probe, allowing for a heat step in the second probe hybridisation without losing the target. Finally the 3’ hairpin is cleaved releasing products for PCR amplification and sequencing that contain only targeted exonic sequences. On-target rates of 97% were reported in their AGBT poster.

2: In their single-probe method a short 50bp probe is hybridised to fragmented gDNA at the 3’ end of an exon, the sequence upstream of this is then enzymatically digested and the 3’ adapter ligated. The probes is then extended to create the complementary strand and a 5’ adapter is ligated to the blunt end. This creates a library with random 5’ ends enabling a duplicate filtering step, unlike PCR approaches.

The protocols are both same-day 6-8 hours with around 1.5 hours hands-on time (according to the posters). Both allow a certain amount of, or all of the off-target sequence to be removed, reducing the amount of sequencing wasted. However the variation in exon length means that some sequence is inevitably lost.

Molecular IDs in cell free DNA: Their single-probe method creates libraries with in-built molecular ID. The random nature of the 5’ end should allow removal of all PCR duplication, without affecting biological duplication too much. Adding a  molecular identifier to the 3’ probe would increase this even further; and also bring molecular ID to the of the dual-probe method.

These molecular ID’s are likely to become increasingly important in methods to call low-frequency mutations in cell-free DNA applications, particularly ctDNA. Current methods make use of deep-sequencing to call mutations just below 1% MAF (mutant allele freq). However simply sequencing deeper may not be enough to get under 0.1%. A MAF of 0.1% would require sequencing to >10,000x to have enough mutant allele reads; and PCR, clustering and sequencing errors all make the detection harder.

Adding a molecular identifier should allow us to develop better statistical methods to call lower and lower MAF. Ultimately we aim to get to a point where we are restricted more by the presence of mutant alleles in a sample than by the technology used to capture and sequence them.

Directed Genomics and cell free DNA: The AGBT poster contained results from the Horizon Diagnostics Multiplex Reference Standard (link). Correlations of observed vs expected allele frequencies were >0.91. This is one of the first methods that can target mutant alleles with a single oligo, as compared to the two used for PCR amplicon sequencing, e.g. TAM-seq. It should mean an increase in sensitivity as more ctDNA molecules can be captured and amplified.

Directed Genomics expects to be launching later in 2015.

2 comments:

  1. Hi James,

    thanks a lot for your summary of their new technology. I am trying to find out more about that company and I was wondering if you can share where you got all that information.
    Thanks a lot!

    Best,
    Roman

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  2. I spoke to the director and founder at AGBT. Here's their details from LinkedIn: https://www.linkedin.com/vsearch/p?company=Directed+Genomics&trk=prof-exp-company-name

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