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  • Pool amplicons on a plate

    I am new to pyrosequencing. I am working on 16s rRNA from environmental samples. I want to measure species diversity, I sequence my fragments from one end of the DNA molecule using the 454 amplicon titanium method.
    One topic in the forum: "Using multiple MIDs in Titanium sequence runs" showed that we can use 10 MIDs-tagged libraries (shotgun samples) in 1 region, but for amplicon sequencing, how many PCR amplicons with diffirent MID-tagged can I pool in 1 region? Can I pool 10 MIDs-tagged in 1 region?
    Last edited by tng012; 07-09-2010, 04:51 AM.

  • #2
    Theoretically the number you can pool would be huge. The question isn't really how many can you run in one region, it's how many should you. The practical limit is mostly due to how many reads you need to obtain from each sample. According to the Roche/454 literature a single large region should produce 360K - 520K high quality amplicon reads (reality is closer to the low end number). How many reads do need to get from each environmental sample to produce an accurate picture of its biodiversity? Divide that number into 360,000 to determine the maximum number of samples you should combine in one large picotiter plate region.

    A very useful resource for microbial 16S rRNA information, and more specifically 454 amplicon sequencing of 16S rRNA can be found through the Ribosomal Database Project. They have a Pyrosequencing Pipeline set up for processing large 16S amplicon data sets obtained from 454 sequencing. Pay special attention to the help page in which they discuss the design of multiplex ID tagged primers. (Note that this page describes primer design for FLX Standard amplicons, not FLX Titanium, but the principles are the same.)

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    • #3
      You could also contact your local Norwegian 454 sequencing center, they might have experience with these kinds of projects. By the way, that would be me... ;-)

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      • #4
        Using regular MIDs "constraints" the number of multiplex to around 155 if I correctly remember it. An other option would be using tags combination in PCR primers. The system we developed allows for 5184 amplicons to be multiplexed. In practice we use around 1152 amplicons in 1/8 PTP plates. The only limit is to make sure that both tags are read during the sequencing as they are both needed for sample assignation. PCR products up to 300bp - 350bp seem fine so far (you can check that with simulating 454 flows on your model sequence).

        The original protocol we developed in the lab is published here :
        Background High-throughput sequencing technologies offer new perspectives for biomedical, agronomical and evolutionary research. Promising progresses now concern the application of these technologies to large-scale studies of genetic variation. Such studies require the genotyping of high numbers of samples. This is theoretically possible using 454 pyrosequencing, which generates billions of base pairs of sequence data. However several challenges arise: first in the attribution of each read produced to its original sample, and second, in bioinformatic analyses to distinguish true from artifactual sequence variation. This pilot study proposes a new application for the 454 GS FLX platform, allowing the individual genotyping of thousands of samples in one run. A probabilistic model has been developed to demonstrate the reliability of this method. Results DNA amplicons from 1,710 rodent samples were individually barcoded using a combination of tags located in forward and reverse primers. Amplicons consisted in 222 bp fragments corresponding to DRB exon 2, a highly polymorphic gene in mammals. A total of 221,789 reads were obtained, of which 153,349 were finally assigned to original samples. Rules based on a probabilistic model and a four-step procedure, were developed to validate sequences and provide a confidence level for each genotype. The method gave promising results, with the genotyping of DRB exon 2 sequences for 1,407 samples from 24 different rodent species and the sequencing of 392 variants in one half of a 454 run. Using replicates, we estimated that the reproducibility of genotyping reached 95%. Conclusions This new approach is a promising alternative to classical methods involving electrophoresis-based techniques for variant separation and cloning-sequencing for sequence determination. The 454 system is less costly and time consuming and may enhance the reliability of genotypes obtained when high numbers of samples are studied. It opens up new perspectives for the study of evolutionary and functional genetics of highly polymorphic genes like major histocompatibility complex genes in vertebrates or loci regulating self-compatibility in plants. Important applications in biomedical research will include the detection of individual variation in disease susceptibility. Similarly, agronomy will benefit from this approach, through the study of genes implicated in productivity or disease susceptibility traits.


        We now use extensions of this protocol with longer tags, allowing for more safety in samples bioinformatics assignation.

        Good luck !

        Jef.

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