Comparing shotgun metagenomic reads: 454 vs HiSeq

[thesoundd]

Hello,

I am currently investigating the bacterial community in the gut of a KO mouse model whose phenotype appears to be connected to its gut microbiota. The bacterial gut community of the KO mouse diverges from that of its WT littermates according to 16S data and we are now trying to understand differences in bacterial functional/gene content that may account for the KO phenotype. I performed a trial of shotgun metagenomics for 3 mice of each genotype and sequenced using 454 FLX technology. I now want to perform shotgun metagenomics on 6 additional mice of each genotype, but I was wondering if I could sequence using Illumina HiSeq technology due to its relatively lower cost. Can you provide feedback on the feasibility of using part-454 and part-HiSeq to develop an understanding of the bacterial metagenome? Thanks!

[TonyBrooks]

Unlocking Short Read Sequencing for Metagenomics

http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0011840

I found this paper a while back. It looks pretty interesting. Still itching to try it out, though. Currently all of our metagenomics stuff is still done on 454 (mostly 16s amplicons).
I think with the new chemistries, you could possibly aim for 300+ bp psuedo read-lenghts with a custom sequencing protocol of 200 cycles, paired end.

[greigite]

We use a similar protocol to the PLoS One paper in that we create metagenomic libraries with narrow insert size distributions, sequence overlapping 100 bp PE reads, and assemble computationally. We did find that size selection with a Pippin prep works better for this than the Ampure selection described in that paper since it provides a narrower size distribution. Using our protocol, we get high quality reads with median size ~150 bp. I plan to try to push this size higher when we start doing 150 bp PE runs.

[wushufa]

http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0011840

I have found a paper describing a strategy for microbiome analysis using 16S rRNA gene sequence analysis on the Illumina sequencing platform. It can produce several Illumina PE100 reads on a single 16S rDNA fragement, so we can get more than 300bp sequences on a 16S rDNA fragment. But the limitation is that 300bp sequences consists 3 100bp-length separated reads.

[wushufa]

As far as I know，v3 region of 16S rDNA can be sequenced by Illumina, and 16S rDNA fragments no more than 240bp can also be sequenced.

[colindaven]

454 is going downhill fast with their inability to increase throughput. The metagenomic shotgun data I have seen from short read sequencers is much better than seen from 454. This is chiefly due to the number of reads obtained. The more reads you get, the better the ability to characterize the functional component of the metagenome.
Analysis can be done with WebMGA, MG-RAST or our approach, Genometa (http://genomics1.mh-hannover.de/genometa/)

See these papers:
Turnbaugh, P. J.; Hamady, M.; Yatsunenko, T.; Cantarel, B. L.; Duncan, A.; Ley, R. E.; Sogin, M. L.; Jones, W. J.; Roe, B. A.; Affourtit, J. P.; Egholm, M.; Henrissat, B.; Heath, A. C.; Knight, R. & Gordon, J. I. A core gut microbiome in obese and lean twins. Nature, Center for Genome Sciences, Washington University School of Medicine, St Louis, Missouri 63108, USA., 2009, 457, 480-484

Qin, J.; Li, R.; Raes, J.; Arumugam, M.; Burgdorf, K. S.; Manichanh, C.; Nielsen, T.; Pons, N.; Levenez, F.; Yamada, T.; Mende, D. R.; Li, J.; Xu, J.; Li, S.; Li, D.; Cao, J.; Wang, B.; Liang, H.; Zheng, H.; Xie, Y.; Tap, J.; Lepage, P.; Bertalan, M.; Batto, J.-M.; Hansen, T.; Paslier, D. L.; Linneberg, A.; Nielsen, H. B.; Pelletier, E.; Renault, P.; Sicheritz-Ponten, T.; Turner, K.; Zhu, H.; Yu, C.; Li, S.; Jian, M.; Zhou, Y.; Li, Y.; Zhang, X.; Li, S.; Qin, N.; Yang, H.; Wang, J.; Brunak, S.; Doré, J.; Guarner, F.; Kristiansen, K.; Pedersen, O.; Parkhill, J.; Weissenbach, J.; Consortium, M. I. T.; Bork, P.; Ehrlich, S. D. & Wang, J. A human gut microbial gene catalogue established by metagenomic sequencing. Nature, BGI-Shenzhen, Shenzhen 518083, China., 2010, 464, 59-65

[smalan]

Sequencing depth for shotgun metagenomic gut microbiome sequencing

Hi
What sequencing depth would enable me to detect microbial species that are present at low levels, when using the shotgun metagenomic sequencing approach in gut microbial samples.

[foofer]

I have the same question Smalan. I'm sure there are some recommendations some where on here but I haven't found them yet.