Difficulty in Host removal by Bowtie2,samtools and bedtools

Question

[priya120195]

hi, I am struggling with host removal step during whole genome shotgun sequencing analysis pipeline of paired end data. When I am running my dataset for mapping with bowtie2 ,It is generating SAM file,it is further ran by samtools.I am getting wrong bam file,because I am getting tructated file in next step of sorting.I really dont know if there is problem with bowtie2 or in samtools.

1. Can anyone suggest a way to check where I am doing wrong.Can we convert SAM file into tabular form to check which reads are mapped and which are not??Is there any script for this?
2. I also wanted to know if there is another tool that can be used for host removal from my WGS paired end datasets. As I am trying to resolve this since 2 months ,still it is not working,If there is another tool for this step then I can try with that.
3. Can we generate blast output from bowtie2??

[Arup Ghosh]

Can you share the commands you are using?

[priya120195]

a) bowtie2 mapping against host sequence Host example: human genome hg19 (download bowtie2 hg19 index)

1) create bowtie2 index database (host_DB) from host reference genome

bowtie2-build host_genome.fna host_DB

2) bowtie2 mapping against host sequence database, keep both mapped and unmapped reads (paired-end reads)

bowtie2 -x host_DB -1 SAMPLE_r1.fastq -2 SAMPLE_r2.fastq -S SAMPLE_mapped_and_unmapped.sam

3) convert file .sam to .bam

samtools view -bS SAMPLE_mapped_and_unmapped.sam > SAMPLE_mapped_and_unmapped.bam

b) filter required unmapped reads
SAMtools SAM-flag filter: get unmapped pairs (both ends unmapped)

samtools view -b -f 12 -F 256 SAMPLE_mapped_and_unmapped.bam > SAMPLE_bothEndsUnmapped.bam

Answer 1

[Antonio R. Franco]

It is sort of hard to understand your questions but if you want to filter your library, take a look to BBSplit

Answer 2

[WouterDeCoster]

1. Can anyone suggest a way to check where I am doing wrong.Can we convert SAM file into tabular form to check which reads are mapped and which are not??Is there any script for this?

SAM is a tabular form. You can just open the file using less or more or head. BAM is binary of SAM, which you can read using samtools view

1. I also wanted to know if there is another tool that can be used for host removal from my WGS paired end datasets. As I am trying to resolve this since 2 months ,still it is not working,If there is another tool for this step then I can try with that.

You'll have to elaborate here about your experiment and what you are trying to solve.

[priya120195]

my experiment is comparative analysis of human gut microbiome of diseased samples.In this regard my pipeline includes quality filtering ,host genome removal,mapping ,analysis of WGS by megan. In the host removal step we are using bowtie2 which generate sam file ,samtools which convert this sam file into bam and generate sorted bam.The problem I am facing is that I am unable to get sorted bam.

[Antonio R. Franco]

I don't see among your scripts any related to the sorting of the sam/bam file

You have samtools sort for that purpose..