Metagenomics with MiSeq

[tonybert]

Greetings, I am new to the super-short read world of Illumina sequencing, and am about to sequence several DNA samples from various soils using the Illumina MiSeq platform (72bp paired end reads, 40 million tags per sample). In previous surveys of marine environments, we used the 454 titanium platform, and I was fairly successful at doing gene-centric analyses (i.e. assessing the distribution of different functional genes/COG categories) with MG-RAST , and other BLAST based tools. I would like to do similar analyses with the soil samples. However, read lengths are much shorter. I was wondering if anyone else has attempted to do such an analysis, and, is there is a way to produce more informative (i.e. longer) nucleotide reads. A secondary goal in this analysis is to do some de-novo assembly, specifically for microorganisms involved in the oxidative components of the nitrogen cycle (ammonia and nitrite oxidizers). If anyone has any tips, or suggestions for how to deal with such data sets, I would appreciate it. Thanks,

[severin]

why 72bp

I am pretty sure the MiSeq can sequence up to 250 paired end so you could in theory get 450-500 base reads by having a short fragment size of say 450-500.

[tonybert]

on 72bp reads

Well, this is the option that was provided by the sequencing center that we are going through. However, at these point they have only constructed the libraries, they have not done any of the sequencing yet. Do you know of an alternative sequencing center that would be able to do such this type of analysis (400 bp) read lengths? Thanks for pointing this out!

[tonybert]

I meant Illumina HiSeq (my apologies)

We are doing sequencing on the HiSeq, no the MiSeq. My apologies.

[pmiguel]

HiSeq (HS) sequence by synthesis (SBS) reagent kits are sold in "50" cycle or "200" cycle units. Both contain enough reagents for all cycles required for index read(s). So, typically one would do one (single read -- SR) or two (paired end -- PE) 51 or 101 base reads on a HS.

My guess is that your core is running older Genome Analyzer (GA) instruments.

Also, despite the Illumina marketing hype, as of right now, there are probably no more than a handful of MiSeq owners able to perform 2x250 base reads. Doing so requires MiSeq v2 chemistry and MiSeq Control Software (MCS) 2.0. The v2 chemistry has been more-or-less available for a week or two. MCS 2.0 is still not. But, I expect it will be by tomorrow.

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Phillip