Combining short and long reads: choosing between PacBioToCA and the new ALLPATHS_LG

After having given an overview of the PacBio error-correction (PacBioToCA) pipeline of Koren et al (see previous blog post), it was interesting to see another paper coming out describing combining PacBio and Illumina for assembling bacterial genomes: Ribeiro et al, “Finished bacterial genomes from shotgun sequence data“, Genome research accepted preprint. The authors are all from the Broad Institute. David Jaffe was so kind as to provide me with the supplementary material file, which so far is not yet available online. In this post, I will summarise the ALPATHS_LG paper, and contrast the approach with the PacBioToCA pipeline.

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The Broad Institute of MIT/Harvard is an impressive genome centre. Sorry, ‘Genomic Medicine Center’. One of their achievements in recent years is an optimised pipeline for assembly of (small and) large genomes based on short read (Illumina) data. The software program they developed, ALLPATHS_LG, combined with a kind of special type of ‘recipe’, has proven to be extremely succesful. The Broad keeps churning out very respectable assemblies of, among other groups, large complex eukaryotes. In order to be able to handle large amounts of genome projcts, I get the impression that the Broad Institute aims for standardisation and optimisation of protocols and, what they call ‘recipes’. The idea being that, if you follow their recipes and use their programs, you are more or less guaranteed an optimal result. In the case of ALLPATHS_LG, it also means, however, that one is required to have at least one Illumina jumping (‘mate pair’) library and a short-insert (‘paired-end’) Illumina library for which the insert size is shorter than twice the read length. Paired end libraries with slightly larger insert sizes are standard, making the ALLPATHS_LG requirements kind of uncommon. It also means not many projects are only able to compare other programs with ALLPATHS without generating an extra read dataset (I speak from experience here…).

The Ribeiro et al paper is entirely focussing on microbial size genomes, a big difference with the Koren et al (PacBioToCA) paper. The short-read recipe for ALLPATHS_LG remains unchanged: 50x coverage short-insert (up to 220bp) paired reads and 50x coverage jumping (2-10kb) reads. This is then supplemented with 50x PacBio reads, with library insert sizes between 1 and 3 kb (although for the paper, they also created two larger insert libraries for two of the strains, at 6 and 10kb respectively). In total, there are three size ranges represented this way: short – overlapping paired reads, intermediate – PacBio reads, long – illumina mate pairs. Finally, the short reads have high quality relative to the PacBio reads.

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Fast genome sequencing of pathogenic bacteria – which benchtop instrument to choose?

Nick Loman was kind enough to give me an advanced copy of his paper in Nature Biotechnology entitled “Performance comparison of benchtop high-throughput sequencing platforms” (Loman et al, 2012). I thought to present a quick summary of the paper here and add some comments of my own.

The paper sets out to “compare the performance of three sequencing platforms [Roche GS Junior, Ion Torrent PGM and Illumina MiSeq] by analysing data with commonly used assembly and analysis pipelines.” To do this, they chose a strain from the outbreak of food-borne illness caused by Shiga-toxin-producing E. coli O104:H4, which caused a lot of trouble in Germany about a year ago. The study is unique in that it is focuses on the use of these instruments for de novo sequencing, not resequencing.

First, they used the ‘big brother’ of the GS Junior, the GS FLX, to generate a reference genome (combining long reads obtained using the GS FLX+, and mate pairs using Titanium chemistry). Then, the same strains were sequenced on the benchtop instruments, and these reads were compared to the reference assembly. The reads were both compared directly, and after assembly with a few commonly used programs.

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More, longer, longest: new reads from three NGS platforms available online

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 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: Those are at Sanger read length, now!

Now I need to find the time to have a look at these data!