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  • Comparing shotgun metagenomic reads: 454 vs HiSeq

    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!

  • #2
    Unlocking Short Read Sequencing for Metagenomics

    Background Different high-throughput nucleic acid sequencing platforms are currently available but a trade-off currently exists between the cost and number of reads that can be generated versus the read length that can be achieved. Methodology/Principal Findings We describe an experimental and computational pipeline yielding millions of reads that can exceed 200 bp with quality scores approaching that of traditional Sanger sequencing. The method combines an automatable gel-less library construction step with paired-end sequencing on a short-read instrument. With appropriately sized library inserts, mate-pair sequences can overlap, and we describe the SHERA software package that joins them to form a longer composite read. Conclusions/Significance This strategy is broadly applicable to sequencing applications that benefit from low-cost high-throughput sequencing, but require longer read lengths. We demonstrate that our approach enables metagenomic analyses using the Illumina Genome Analyzer, with low error rates, and at a fraction of the cost of pyrosequencing.


    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.

    Comment


    • #3
      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.

      Comment


      • #4
        Background Different high-throughput nucleic acid sequencing platforms are currently available but a trade-off currently exists between the cost and number of reads that can be generated versus the read length that can be achieved. Methodology/Principal Findings We describe an experimental and computational pipeline yielding millions of reads that can exceed 200 bp with quality scores approaching that of traditional Sanger sequencing. The method combines an automatable gel-less library construction step with paired-end sequencing on a short-read instrument. With appropriately sized library inserts, mate-pair sequences can overlap, and we describe the SHERA software package that joins them to form a longer composite read. Conclusions/Significance This strategy is broadly applicable to sequencing applications that benefit from low-cost high-throughput sequencing, but require longer read lengths. We demonstrate that our approach enables metagenomic analyses using the Illumina Genome Analyzer, with low error rates, and at a fraction of the cost of pyrosequencing.

        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.

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        • #5
          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.

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          • #6
            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

            Comment


            • #7
              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.

              Comment


              • #8
                I have the same question Smalan. I'm sure there are some recommendations some where on here but I haven't found them yet.

                Comment

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