As before, full run throughput in gigabases (billion bases) is plotted against single-end read length for the different sequencing platforms, both on a log scale. Yes, I know a certain new instrument seems to be missing, hang on, I’m coming back to that…
With this post I present a figure I’ve been working on for a while now. With it, I try to summarise the developments in (next generation) sequencing, or at least a few aspects of it. I’ve been digging around the internet to find the throughput metrics for the different platforms since their first instrument version came out. I’ve summarised my findings in the table at the end of this post. Then, I visualised the results by plotting throughput in raw bases versus read length in the graph below.
A potential user (‘customer’) of our sequencing platform asked how to generate reference genomes for his 4 bacterial strains. His question inspired me to write this post. The suggestions below are not absolute, just my thoughts on how one these days could go about sequencing a bacterial genome using one or more of the sequencing platforms. I would appreciate any feedback/suggestions in the comments section!
Option 1: bits and pieces
- Libraries: paired end or single end sequencing
- Platform: one or more of Illumina MiSeq or HiSeq, Ion Torrent PGM, 454 GS FLX or GS Junior
- Bioinformatics: assembly: Velvet, SOAPdenovo, Newbler, MIRA, Celera
- Outcome: up to hundreds of short contigs (with only single-end reads) or contigs + scaffolds (with paired end reads)
- Pros: fast and cheap, OK for presence/absence of e.g. genes
- Cons: doesn’t give much insight into the genome
- Remarks: due to per-run throughput, multiplexing is recommended; data can also be used for mapping against a reference genome instead
I am not attending the American Society of Human Genetics meeting in San Fransisco, but can’t escape the buzz it creates on twitter (hashtag #ashg2012). Strikingly, it is almost another AGBT when it comes to announcements from companies selling sequencing instrument. All of them had something new to bring to the floor. This post summarizes what I picked up from twitter and a few websites, and I give a bit of my perspectives on the respective announcements. I am focussing on technology improvements, especially with regard to read lengths, not so much on applications such as cancer resequencing panels.
Supposedly, if you need long reads of high quality and throughput, you should be using GS FLX+, yielding 750-800 peak read length, from Roche/454, right? Illumina reads are getting longer, but 250 is for now the limit (notwithstanding the 300 bp run done by the broad). IonTorrent promises 400 bases, but we will have to see what the quality is going to be. And PacBio, well we all know they are long, but with relatively low throughut and quality peaking at 85% – 86% accuracy (useful nonetheless).
Commercially, GS FX+ had been around for more than half a year. So far, the community is reporting some success, see this thread at SeqAnswers. But, there are problems all around when you talk to people. Our own centre (the Norwegian Sequencing Centre) got the upgrade in August. I’ll spare you the details on numerous control/test fragment runs, short read runs etc, but the bottom line is that we still haven’t been able to sucessfully sequence a FLX+ library on our GS FLX+. Right now, we are having a service visit, and I intend to chain the guy to the instrument until we see some real good data from one of our own libraries…
There is another thing that surprises me too: I am attending PAGXX, the Plant and Animal Genome conference in San Diego, where Roche is one of the main sponsors. However, when you look at the little text they have as an exhibitor, well, let me just quote a part of it for you (source): Continue reading
For three platforms, reads longer than the commercially available, and/or from not-yet released instruments, have become accessible online. With online, I mean that we all can download these data to have a look at:
1) MiSeq 2x 150 bases runs
As part of the German E. Coli (EHEC) ‘Crowdsourcing Project’, Illumina sequenced fie strains for the UK Health Protection Agency, the fastq files can be downloaded from http://www.hpa-bioinformatics.org.uk/lgp/genomes. These are the first data in the public domain from a MiSeq!
See also this post on GenomeWeb.
2) IonTorrent 316 chip
Keith Robison shares a bit of info on data from an Ion 316 chip ion his ‘Omics! Omic!’ blog: “1.69M reads, with 1.53M of those >=50 bp long and 1.07M 100bp or longer”:
I downloaded the run files, and quickly looked at the read length distribution of the trimmed reads in the sff file (which listed 260 flows, 40 more than the file I analyzed in my previous post), showing a peak exactly one base longer at 109 bases. So, many more reads but not much gain in length (yet). Note the strange shape of the peak:
3) 454 GS FLX+
As part of the assemblathon2 (a de novo assembly competition), there have been released the first GS FLX+ reads (from a parrot), peak read length around 736 bases: http://bioshare.bioinformatics.ucdavis.edu/Data/hcbxz0i7kg/Parrot/. Those are at Sanger read length, now!
Now I need to find the time to have a look at these data!