unmapped BAM from Ion 16s Metagenomics Kit using MergeBamAlignment

osaama_sosaama_s Member
edited February 21 in Ask the GATK team

I have sequenced 16s rRNA from waste water samples using Ion 16s Metagenomics kit. After paired-end sequencing using the machine Ion S5, I have received unmapped BAM files for all samples. Each uBAM contains both forward and reverse reads unaligned. I want to map those unmapped BAM files (uBam --> Bam) using the reference genome (16s gene) before I can convert them to fastq for analysis in QIIME 2.0. However, I am stuck at how to map the ubams? I am new to bioinformatics so any help will be appreciated! Do I have to sort uBAMs in some order before mapping them? I have installed picards but I am not sure which plugin should I sure to get fastq files which I want in the end for QIIME analysis.

Best Answer

Answers

  • SkyWarriorSkyWarrior TurkeyMember
    edited February 21

    You need to use PICARD SamToFastq to convert your Ubam to fastq. Check picard tools page for more details. Normally if you want to start mapping directly from ubam you need to pipe SamToFastq to your mapper of choice.

  • @SkyWarrior thank you but the main problem here is not to convert Ubam to fastq. I have raw paired-end sequence data from Ion Torrent in UBAM file. How do i map it to a reference genome to get a mapped BAM file which can then be later to converted to fastq?

  • SkyWarriorSkyWarrior TurkeyMember

    I really don't know what to say... :|

  • SheilaSheila Broad InstituteMember, Broadie, Moderator

    @osaama_s
    Hi,

    I hope I have answered in this thread.

    -Sheila

  • osaama_sosaama_s Member

    @Sheila

    No, please see my response to the thread where you suggested the tutorial.

  • osaama_sosaama_s Member

    @SkyWarrior this was VERY helpful! I really needed this sort of information. Thank you for this! This clears many troubles. I have tried converting uBAM --> FASTQ using samtools and picard (using the command you suggested) but it gives me one output fastq file which has the reads and the second fastq file generated by picard, however, is an empty file. My sequencer people, however, confirm that uBAM they gave me consists of both forward and reverse reads but unaligned. Therefore, how do I get them out of UBAM?

  • SkyWarriorSkyWarrior TurkeyMember

    That is another issue. It is possible that uBAM is consists of only single end reads. In that case there is really nothing that you can do about it. ION kits mostly do single end sequencing but only some special kits that do very long reads have paired end solution. Sequencing people may be misguiding you as well. You may ask them to provide you the file in fastq format if possible. Although ION is widely used platform for sequencing, it is not the preferred one by many tool builders in NGS field.

  • SheilaSheila Broad InstituteMember, Broadie, Moderator

    @osaama_s
    Hi again,

    I think section 3 of this tutorial will help.

    -Sheila

Sign In or Register to comment.