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preprocessing raw sequence data in a UBAM file from IonTorrent 16S Metagenomics kit

Hi everyone!

I have raw, paired-end sequence data in a UBAM (unmapped) file after sequencing 16S gene from water samples. The UBAM file contains both forward and reverse reads but unjoined. How can I do the following:

1) Map UBAM to a reference genome so that I will have an aligned, paired-end mapped BAM file? I heard you can use picard but which function to use, I am very unsure.

2) Prepare that BAM file for analysis in QIIME 2.0 by converting it to fastq? I believe I can use samtools to convert BAM to FASTQ but I want to get your advice.

These may be silly questions but I am new to bioinformatics so any help will be appreciated!

Answers

  • SheilaSheila Broad InstituteMember, Broadie, Moderator admin

    @osaama_s
    Hi,

    1) I think this tutorial will help.

    2) You can use BamToBfq.

    -Sheila

  • osaama_sosaama_s Member

    @Sheila thank you for your response! The tutorial you suggested in going in opposite direction. It is guiding on how to get a uBAM from BAM or FASTQ (I do not want that).

    My problem is different: the end goal of the project is to get a fastq file ready for analysis in QIIME 2.0.

    Therefore, I am trying to figure out a way to process the raw uBAM file (which contains unaligned paired end data). What I need to know is how to align the paired-end reads in the raw uBAM file to a reference genome, getting an aligned BAM file in the end?

  • SheilaSheila Broad InstituteMember, Broadie, Moderator admin
    edited March 5

    @osaama_s
    Hi,

    The tutorial does guide you on that, but you can also use it to align your uBAM with BWA. Note, you need to convert your uBAM to FASTQ to run BWA. Section 3 helps with that. You will get a clean aligned BAM from following that tutorial. Also note, you can start with section 2 since you are starting with uBAM. From the tutorial: "If you have raw reads data in BAM format with appropriately assigned read group fields, then you can start with step 2."

    -Sheila

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