NATURE BIOTECHNOLOGY | Performance comparison of benchtop high-throughput sequencing platforms

NATURE BIOTECHNOLOGY | RESEARCH | ANALYSIS

Performance comparison of benchtop high-throughput sequencing platforms

• Nicholas J Loman,
• Raju V Misra,
• Timothy J Dallman,
• Chrystala Constantinidou,
• Saheer E Gharbia,
• John Wain
• & Mark J Pallen

• Affiliations
• Contributions
• Corresponding authors

Nature Biotechnology

 

30,

 

434–439

 

(2012)

 

doi:10.1038/nbt.2198

Received

 

19 December 2011 

Accepted

 

30 March 2012 

Published online

 

22 April 2012 

Corrected online

 

23 April 2012

Abstract

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• Supplementary information

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Three benchtop high-throughput sequencing instruments are now available. The 454 GS Junior (Roche), MiSeq (Illumina) and Ion Torrent PGM (Life Technologies) are laser-printer sized and offer modest set-up and running costs. Each instrument can generate data required for a draft bacterial genome sequence in days, making them attractive for identifying and characterizing pathogens in the clinical setting. We compared the performance of these instruments by sequencing an isolate of Escherichia coli O104:H4, which caused an outbreak of food poisoning in Germany in 2011. The MiSeq had the highest throughput per run (1.6 Gb/run, 60 Mb/h) and lowest error rates. The 454 GS Junior generated the longest reads (up to 600 bases) and most contiguous assemblies but had the lowest throughput (70 Mb/run, 9 Mb/h). Run in 100-bp mode, the Ion Torrent PGM had the highest throughput (80–100 Mb/h). Unlike the MiSeq, the Ion Torrent PGM and 454 GS Junior both produced homopolymer-associated indel errors (1.5 and 0.38 errors per 100 bases, respectively).

Figures at a glance

left

Torrent Suite Software Mobile Browser

Neat!!

Working with Reference Genomes in Torrent Suite Software 1.4

Nick Loman blogs PGM 316 1st Impressions

 While eagerly waiting for our own PGM to be installed for 316 chip support, I came across Nick's review on their own 316 run. Which has pretty impressive output! 

"Our first two runs of 316 chips yielded an impressive 251Mb and 209Mb respectively! Mean read length was about 110bp."

Other interesting factoids for the impatient 

we've loaded the chips way higher than we are used to with the 314 – densities of 76-82%

This reflects a change to the protocol – when we were running 314 chips we were told to load fewer beads to get better coverage – and from our trials when we loaded at 41, 43 and 46% density on the 314 chip the 41% run did do best. The 314 chip has about 1.2m wells, so we were filling about 550k wells. About two-thirds of those wells were live spheres (meaning they have DNA on them) and out of those about two-thirds pass the quality filter – about 200k reads in all (~20Mb data).

The 316 chip has 6.3m wells and we're filling about 5m of these. A little under half are passing the quality filters, meaning we're getting about 2.25m reads.

 

Do hop on over to the original post to see FASTQC plots of the reads

Ion Torrent 316 First Impressions

By Nick Loman on July 28, 2011

:) Ion Torrent Server up!

RNA-seq on the Ion Torrent PGM

K I must admit with the 314 chip, 500k reads seem .... stretching the limits of usability for RNA-seq. but looking at the Life Tech's presentation New to RNA-seq; how it compares to microarrays. it does makes sense to use PGM over microarray for certain reasons.. and certain samples. e.g. bacteria / virus transcriptomes. Granted that PGM also gives better dynamic range than microarrays with a price that's not too far from microarray, it does make sense to beef up one's data with a run or two of Ion Torrent.

at USD $595 for a 316 chip run "All included"as quoted. They do make it very attractive for microarray users to switch over. Granted u might need a couple of runs to make sense of human samples.
Though I be wary about hidden costs not anticipated in their calculations.
What's interesting is that they claim no platform bias between SOLiD and PGM runs, no details are given, but i assume they ran PGM runs to match SOLiD Throughput and compared the output?

Would you consider PGM for ur RNA-seq?
post in comments please...

A 3rd party evaluation of Ion Torrent's 316 chip data

Dan Koboldt (from massgenomics) has posted about what I know to be the 1st independent look at the data from Ion Torrent's 316 chip,
Granted the data was handed to him in a 'shiny report with color images' but he has bravely ignored that to give an honest look at the raw data itself.

The 316 chip gives a throughout that  nicely covers WGS reseq experiments for bacterial sized genomes. "The E. coli reference genome totals about 4.69 Mbp. With 175 Mbp of data, the theoretical coverage is around 37.5-fold across the E. coli genome."

For those wary of dry reviews, fear not, easily comprehensible graphs are posted within!

read the full post here

Ion Torrent, de novo assembly and a nasty bug

Nick Loman's blog post looking at de novo assembly with Ion Torrent did over 2,000 views within a short time, attesting to the interest that everyone has with de novo assembly or Ion Torrent data,

His post

Ion Torrent data blog post; a week is a long time in genomics

also touches on N50 values, a common 'metric' for de novo assembly

CLC Bio wrote a press release
from his blog post

However, he also pointed out MIRA as a excellent free assembler..

Webinar Introduction to Ion Torrent Informatics

Webinar by Life Technologies
Introduction to Ion Torrent Informatics
4 May 2011
Register via this web link

Event Title

Introduction to Ion Torrent Informatics

Event Description

The Ion Torrent semiconductor sequencing platform includes a preconfigured Torrent Server that processes data from the Ion PGM™ Sequencer. With each semiconductor sequencing run, data analysis occurs on the Torrent Server. Scientists can interact with Torrent Server through the remotely accessible Torrent Browser web interface. Both processing status and run performance are easily viewable through these web pages. From Torrent Browser, detailed analysis reports can be viewed or sequencing data can be downloaded to your local computer for downstream analysis. The Torrent Suite Software formats base call and alignment data using industry standard data formats giving users the flexibility to use a wide variety of analysis tools. For scientists who are looking for analysis solutions, several downstream software packages are available and will be briefly demonstrated to show how DNA variations can be identified.

What do I do with a PGM?

I am curious if 1 Gbp is enough for broad views of metagenomics, cursory glances of WGS for eukaryotes, and targetted sequence capture..  here's views by others on how they would use the PGM

IonTorrent: Benchtop Sequencing, Streamlined

from MassGenomics by Dan Koboldt

PGM Applications

It’s a good thing to see the PGM constantly evolving - first the throughput was doubled, and now sample prep time cut substantially. At the price points we’re talking about, this might easily become standard equipment or small labs, academic departments, even single investigators. The current throughput of 1 Gbp isn’t enough for whole-genome or whole-exome sequencing, but it opens the door to a number of targeted applications. In Genome Technology’s Cancer Issue this month, for example, I read about a group that’s using the PGM for a clinical test comprising 100 common mutations in human cancers.
Essentially, the niche for PGM, MiSeq, and GS Junior is everything that’s not quite enough for a full-on sequencing run. A few examples come to mind:

1. Microbial sequencing. For bite-sized genomes, 1 Gbp should be more than enough. Imagine walking into a clinic to have your strain of Streptococcus or some other infection sequenced the same day.
2. Family linkage studies. With a few family members and a reasonably-sized linkage peak, you could sequence all gene-coding exons across a region of interest, either by PCR or custom capture.
3. Orthogonal validation. Whole-genome and whole-exome studies might identify hundreds of putative mutations. Emphasis on putative. No matter how good your algorithms and filters are, there will be some false positives. Here’s an opportunity for a small, fast validation instrument. Preferably, you choose a different sequencing technology for validation (e.g. PGM for Illumina, MiSeq for SOLiD).

For more, see Keith Robison’s post at Omics! Omics! or Matthew Herber’s blog on Forbes.com.

Softgenetics Nextgene has Ion Torrent analysis webinar

NextGENe 2nd generation sequence analysis

Update: links are javascripted popups (hate those, tried inserting the header if it still doesn't work , you need to go via
http://www.softgenetics.com/analysisCorner2.html

Thanks for the headsup!

NextGENe Software Basics for Biologists
http://www.softgenetics.com/webinarC.html

Target Capture Analysis with NextGENe (part 1)
http://www.softgenetics.com/webinarA.html

Target Capture Analysis with NextGENe (part 2)
http://www.softgenetics.com/webinarB.html

Ion Torrent
http://www.softgenetics.com/webinar/sg_iontorrent.html