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  • Pipeline construction (metagenomics 101)

    Hello guys / gals,
    I really feel that this might be one of the last options and almost a cry for help, I've been reading and studying metagenomics field for over a month now, and I still have a not properly understood the concepts of it. If you would help me or guide me in any way into a general direction of better understanding it i would be extremely grateful.

    So here is the problem, I am a master student who's new to bioinformatics field (had only 1 lecture of it) and now I am assigned to make a pipeline for metagenomic analysis. There are some main ideas that are expected of me, and I've been reading literature on it, but I find it really hard to review this literature as I do not fully understand them and also I have never written a thing like that.

    So the objective is to propose a pipeline, in which sampled data would be placed into phylogenetic trees to see who is in the sample in reference to some refence database. The input is FASTA files from metagenomic bins ( there's a pipeline for sorting them into contigs, filtering , quality control and etc. ) which is expected to be a single genome in a single bin. From this place I should take over and suggest tools for further data processing. The steps in my pipeline should be:

    1) Selecting Marker genes
    2) Indentifying them in the bins
    3) Allignment processing
    4) Concatenation of marker genes
    5) Applyig evolutionary models and
    6) infering phylogenetic trees.


    So here goes some questions about it, and sorry if the answers are obvious to you (because they are not for me..) :

    I've seen in literature that Amphora(2) and Phylophlan are the methods using concatenated marker gene phylogeny, but others (most that i found) use OTUs that seem to be used as the same thing, is there a difference between them?

    I try to refer to the literature I am reading, but there is too much variation and I do not feel confident that they are answering the same questions I should be. Is there any good/ working pipelines that are working on the same or similar protocol that i described?

    How should i choose the reference genomes and reference marker genes, as I cannot see any correlation between different literature, maybe you have any information where I could read it up?

    If you have any comprehensive literature that might be helpful on any of the steps (with full explanations of what is going on in each step), could you share it?


    TL; DR

    rookie at bioinformatics requests for help in building a pipeline, all help would be appreciated.

    Thank you for your time

  • #2
    Check this: http://ab.inf.uni-tuebingen.de/software/megan6/
    It uses DIAMOND to align sequences, then MEGAN for the taxonomic analysis.
    Hope it helps!

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