Extracting or aligning forward and reverse reads from ubam file

Question

[osamashehzad.786]

I am performing microbiome analysis in waste water using Ion 16s Metagenomics kit. I have .ubam files which I have successfully converted to .fastq using the following option in samtools

samtools fastq -0 out.fastq input.ubam

Should I have used the -0 option from samtools?

The original ubam file contains paired-end reads (both forward and reverse reads, un-joined within the file). Also, they have barcodes and adaptors attached to the sequences within the .ubam file. How can I accomplish the following goals:

1) get both forward and reverse reads out

2) join them (i will use qiime 2.0 for it).

Also, I used IonXpress 1-16 barcodes but I cannot find the sequences of the barcodes which are required for demultiplexing. I spent an hour looking at their website but I cannot find the sequences. Can anyone help me please?

Here is the how the .fastq files look likes.

@GLPH7:00666:05788
GGACTACCAGGGTATCTAATCCTGTTTGCTCCCCACGCTTTCGCATCTCAGCGTCAATAATGTTCCAGTAGATCGCCTTCGCAATCGGTATTCCTTCTGATCTCTACGGATTTTACCCCTACACC
+
A<BCBBC?ABD=CC:;:B=AB@;;;B0000000+00000>8@>>>?@@A:9:99@@<>>:0005:5008;99;://)/6///;5://*/99;?<B9<839==55599<7::::+4///&/7///1
@GLPH7:00666:05802
GCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGCCATTAGTTGCCATCATTAAGTTGGGCACTTTAATGGGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAGTCCTCATGGCCCTTATGACCAGGGCTTCACACGTCATACAATGGTCGGTACAGAGGGTTGCCAAGCCGCGAGGTGGAGCTAATCTCATAAAACCGATCGTAGTCCGGATCGCAGTCTGCAACTCGACTGCGTGAAGTCGAAATCG
+
;:>==999@9;:;<;<AABA@=>>>>@@@CC>CID@CBBC>CBA=@??>999A?BD=C>@A@BB>@@@899:@9:9@?C;BA>BB;B@@@C=C?CCG>AA9::?B=BA@>>>:?=A;??=A:@;?@@@09::>A@@AB@GDD?BBBBB@DD>C=BBBBD@CBB<AA@BBAAABA@?BB==;=A>A<=;<AA<>>BD>C?B>:A489>=@@@@<@A=@@@@@<@>>>?====2=9///;;:?===7<6::///9:;>>>===B<;;AA>>>A;;;5:=888B;?;<

Answer 1

[h.mon]

You may try to follow the easy and clear instruction from one of several product manuals:

Barcode (A) adapter sequences are available on the Ion Community. Visit the Ion Community at ioncommunity.thermofisher.com and perform a search for "Ion 96 Barcode Set."

If you do that, you will NOT find the adapter sequences, though.

So you may download them from the Batzer Lab (an excel sheet with the 1-96 barcodes), or you may install the Torrent Suite software, as you can view the barcode sets and the barcode sequences for the pre-installed Ion Torrent barcodes. Maybe you can get the reads out of the uBAM with the Torrent Suite as well.

[osamashehzad.786]

Thank you for this! :)

Also, do you have any idea how can I extract forward and reverse reads in .fastq files from a single UBAM using samtools? The original UBAM contains both forward and reverse reads but unaligned.

[h.mon]

There are several ways of extracting the reads from a bam file. I never tried with an uBAM file, but they should work.

With samtools, you can use samtools fastq (might be good to do samtools collate | samtools fastq). I believe samtools bam2fq is synonymous to samtools fastq since SAMtools 1.3. You also can use htscmd bam2fq from HTSlib.

You can also use bam bam2FastQ from bamUtil.

Or bedtools bamtofastq from bedtools.

Or reformat.sh from BBTools.