Thursday, 20 November 2014

Copy-number: exomes vs genomes, proposing a better strategy

Comparing CNVs
Exome sequencing (@Exome_seq) can be used to generate CNV maps bit the data are noisy compared to WGS or SNP-arrays. In this post I’ll describe a novel workflow to get high-quality CNV from an exome-seq pipeline and show some very new data…

Image reproduced and enlarged at the bottom of this post.

Monday, 17 November 2014

Ion Proton amplicons for clinical molecular diagnostics

A recent paper from the MD Anderson's Department of Hematopathology reports on their use of the AmpliSeq for a 409 gene panel on Ion Proton: Clinical massively parallel next-generation sequencing analysis of 409 cancer-related genes for mutations and copy number variations in solid tumours. I'm a big fan of amplicomes, another 'ome I know, and in this context used to mean all the amplicons in your panel. PCR is a great way to enrich for specific regions of the genome and we all understand the basics. For me amplicomes on NGS is perhaps the easiest NGS for the NHS. It simply replaces M13-PCR for a single amplicon with a multiplex PCR, and Sanger sequencing with NGS. If a doctor understood a Sanger test then this is perhaps an easier leap to make than trying to understand/explain what an exome is!

Back to the paper:  The MD Anderson group used their gene panel on FFPE material and reported on the "sensitivity, specificity, reproducibility, and applicability of using the Ion Proton 409-gene panel to routinely screen for SNVs, insertions/deletions, and CNVs". To do this they used 55 tumours (20 with paired normals) and four cell lines.

Fig 6A: ERBB2 amplifications as seen in 4 breast cancer samples.

The Ion Torrent Ampliseq Comprehensive Cancer Panel uses 4000 primer pairs across four primer pools each requiring just 15ng on input DNA (60ng total), and up to 96 samples can be indexed using Ion Xpress Barcodes. Alignment and analysis of data was performed using the Torrent Suite software. And the groups OncoSeek was used to generate a clinical report.

Concordance between platforms was high when the group compared results to an earlier PGM panel of 46 genes, and detected pretty much everything they expected. The Proton did call some InDels that the PGM missed, which the group put down to improvements in calling software.

The group reported high sensitivity for SNVs up to 5% allelic fraction, and high reproducibility. This, coupled to the minimal FFPE input, and fast turnaround (5 days) makes the platform combination one that could be used in a clinical molecular diagnostics laboratory. Additionally they reported that up to 10 samples could be multiplexed per run and that this fitted in particularly well with their workflows.

Friday, 14 November 2014

Supercentenarian genomes: but are they the right ones

GenomeWeb covered a recent paper from Stuart Kim's group at Stanford University: Whole-Genome Sequencing of the World’s Oldest People. Unfortunately they did not find any longevity genes, and Kim was quoted saying "We were pretty disappointed."

There is lots of suggestion that longevity has a genetic component, but I can't help but consider that the environmental component is likely to be stronger, and this would mask the genetics. Kim's study was very small, just 17 genomes, so the chances of finding anything were equally small. But had there been low-hanging fruit this would almost certainly have been a Science paper rather than PLoS One (sorry PLoS).

Monday, 10 November 2014

HiSeq X Ten: when might they be available one at a time?

In the Summer I posted a Christmas letter to Santa. He's already delivered number 2 (cheaper RNA-seq) and 3 (longer RNA-seq reads via PE250), and number 4 might be coming soon (exomes at PE125). I'd also asked that he not deliver HiSeq X Ten as an individual instrument just yet, but as there are just 44 days left till Christmas I thought I'd look head and see what might be the reasons for, or not for gift wrapping a single X Ten this Christmas.

Friday, 7 November 2014

ctDNA in triple negative breast cancer: a study comparing Illumina & 454 sequencing

A recent circulating tumour DNA (ctDNA) paper describes a comparison of ctDNA to CTC's with respect to TP53 mutations in 40 triple negative breast cancer patients. See: Circulating tumour DNA and circulating tumour cells in metastatic triple negative breast cancer patients in the International Journal of Cancer.

Wednesday, 5 November 2014

250bp paired-end on your HiSeq 2500...up yours X Ten ;-)

The 250bp paired-end HiSeq rapid run kits are almost here! Expect to get 150M reads per lane or more on the current rapid flowcells. What will you do with 150M 500bp OPES?

No word on pricing yet, and I could not find any PE250 data on BaseSpace. Looking forward to finding out more...

Friday, 17 October 2014

Your own personal genome project

I was chatting with a colleague at work who'd asked me if I know anywhere they could get their genome or exome sequenced. My genome has been sat in the freezer for over five years wanting to go onto a flow cell, but I've never been comfortable putting it on our own machines. I did get 23andMe'd a few years ago but they've closed the exome for now.

Today there are many sequencing service providers across the world. Would any of them be open to a consumer led project? How many genomes/exomes would we need to sequence to get a price consumers were willing to pay? To test the market we've used AllSeq: "the global sequencing marketplace", and a couple of replies have now come in!

Thursday, 9 October 2014

Twitterbots for NGS

I've been inspired to create three Twitterbots for NGS papers on @RNA-seq, @ChIP-seq and @Exome-seq by Casey Bergman at the University of Manchester. I'd not come across Caseys twitter account (I don't actually use Twitter that much) or his lab website and blog; but I was directed there by a piece on the Nature website...How to tame the flood of literature.

What Casey has done is pretty simple and it is very well explained in his blog post, or by Rob Lanfear who has posted instructions on GitHub. There are three simple steps for PubMed and Twitter (and more for arxiv, peerj, etc).
  1. Set up a twitter account
  2. Set up a pubmed search
  3. Set up your account
The feeds I created only went live this evening and I'll follow Robs advice to refine them over the next few weeks. Let me know if you like them, and why not create your own feed.

PS: Casey has a great post on how to host a custom UCSC genome browser trackwith Dropbox.



Tuesday, 7 October 2014

Count-down to AGBT 2015

Registration is open! The registration process for AGBT has been overhauled in the last few years; yes there's still a lot of people who don't get in, but it seems to be pretty fair. 

I hope to see you there. I'm going to be blogging and Tweeting again (assuming I get in). Say Hi if you read CoreGenomics and I'll buy you a beer (or rather grab one from Illumina, Agilent, etc)!

Thursday, 2 October 2014

Whole exomes from single cells: Fludigm C1 update

This was not a planned post but it follows on nicely from today's other one about exomes. This time I'm writing about Fluidigm's new single-cell exome-seq protocol. Yup that's right, whole exomes from 96 single cells! The C1 is an amazing piece of kit (wish I had one) and we've used it a little bit for mRNA-seq. The ability to sequence single-cell genomes and exomes means you can pretty much do whatever you want with a single-cell now. So how do the exomes look?