FASTA sequence From large BAM file

mez · 11-12-2011, 03:38 PM

I hope I would be able to get some help even though my question will sound very basic, I am new to sequencing formats.

I am working on a project that involves obtaining reference sequences from the .bam files in the 1000 genomes database, to perform disease studies for a group. I used samtools view to download the genomic region I am interested in, but the actual sequence column in the .bam files seem to have reads for both forward and reverse strands. I would like to know, how I could get the simple nucleotide sequence of the reference strand from .bam file ... some kind of consensus sequence I am guessing.

maubp · 11-13-2011, 01:01 PM

Are you trying to get the consensus sequence from a BAM file?

The reference sequence would be the same standard human genome used for all the 1000 genomes, and can probably be downloaded from their site as FASTA.

mez · 11-13-2011, 02:50 PM

Yes, I want to get the consensus FASTA sequence from the BAM file for a given chromosome region(not necessarily the whole chromosome sequence).

maubp · 11-14-2011, 04:34 AM

Have you tried searching the forum (or Google) for the terms BAM consensus?

Look at the samtools mpileup command (which replaced the older samtools pileup command).

ardmore · 11-14-2011, 10:13 AM

I have the same question. I used the command

Code:

samtools mpileup -uf ref.fa aln.bam | bcftools view -cg - | vcfutils.pl vcf2fq > cns.fq

However I got an error.

Code:

the 'Argument “f” isn’t numeric in numeric

maubp · 11-14-2011, 02:12 PM

You're not the only person to hit that,
http://biostar.stackexchange.com/que...ric-in-numeric

ardmore · 11-14-2011, 03:18 PM

Well. I think that it is the bug of samtools manual.
I used the command

Code:

samtools pileup -cf ref.fa aln.bam | samtools.pl pileup2fq -D100 > cns.fastq

Everything is fine.

swbarnes2 · 11-14-2011, 06:35 PM

Quote:

Originally Posted by ardmore

Well. I think that it is the bug of samtools manual.
I used the command

Code:

samtools pileup -cf ref.fa aln.bam | samtools.pl pileup2fq -D100 > cns.fastq

Everything is fine.

Pileup is deprecated. Everyone uses mpileup. Don't expect a lot of help if you stay with pileup.

And you do realize that neither method will touch putative indels, right?

maubp · 11-15-2011, 01:27 AM

Quote:

Originally Posted by swbarnes2

And you do realize that neither method will touch putative indels, right?

What do you recommend for getting an indel aware consensus from a BAM file?

kriikku · 01-13-2013, 06:42 AM

See here for one way to get a .vcf file with SNPs and indels from the .bam file, or a consensus sequence:
http://samtools.sourceforge.net/mpileup.shtml

The consensus sequence generated by this method has the problem that it only applies the SNPs to the reference sequence, but not the indels.
The .vcf file is better since it includes both SNPs and indels.

The .vcf file can be converted to a .fasta sequence using this tool:
https://www.broadinstitute.org/gatk/...Reference.html
However, note that this tool will only take into account indels of length up to 2 bases (as of January 2013). You may want to write your own script to insert all the indels (including the longer ones) from the .vcf into the .fasta.

Similar Threads
Thread	Thread Starter	Forum	Replies	Last Post
Extract sequence from multi fasta file with PERL	andreitudor	Bioinformatics	27	07-07-2019 08:45 AM
introducing BAMseek, a large file viewer for BAM and SAM	BAMseek	Bioinformatics	11	07-23-2013 09:02 PM
Find all occurrences of a sequence in a fasta file	dphansti	Bioinformatics	3	12-06-2011 07:11 AM
To get the sequence from a given BAM file	ardmore	Bioinformatics	11	11-12-2011 11:22 AM
Bam and Sam don't like my fasta file	mindlessbrain	Bioinformatics	2	12-09-2010 11:47 PM

11-12-2011, 03:38 PM	#1
mez Junior Member Location: georgia Join Date: Nov 2011 Posts: 5	FASTA sequence From large BAM file I hope I would be able to get some help even though my question will sound very basic, I am new to sequencing formats. I am working on a project that involves obtaining reference sequences from the .bam files in the 1000 genomes database, to perform disease studies for a group. I used samtools view to download the genomic region I am interested in, but the actual sequence column in the .bam files seem to have reads for both forward and reverse strands. I would like to know, how I could get the simple nucleotide sequence of the reference strand from .bam file ... some kind of consensus sequence I am guessing.

11-14-2011, 10:13 AM	#5
ardmore Member Location: USA Join Date: Jun 2011 Posts: 51	I have the same question. I used the command Code: samtools mpileup -uf ref.fa aln.bam \| bcftools view -cg - \| vcfutils.pl vcf2fq > cns.fq However I got an error. Code: the 'Argument “f” isn’t numeric in numeric

11-14-2011, 03:18 PM	#7
ardmore Member Location: USA Join Date: Jun 2011 Posts: 51	Well. I think that it is the bug of samtools manual. I used the command Code: samtools pileup -cf ref.fa aln.bam \| samtools.pl pileup2fq -D100 > cns.fastq Everything is fine.

01-13-2013, 06:42 AM	#10
kriikku Junior Member Location: Estonia Join Date: Jan 2013 Posts: 5	See here for one way to get a .vcf file with SNPs and indels from the .bam file, or a consensus sequence: http://samtools.sourceforge.net/mpileup.shtml The consensus sequence generated by this method has the problem that it only applies the SNPs to the reference sequence, but not the indels. The .vcf file is better since it includes both SNPs and indels. The .vcf file can be converted to a .fasta sequence using this tool: https://www.broadinstitute.org/gatk/...Reference.html However, note that this tool will only take into account indels of length up to 2 bases (as of January 2013). You may want to write your own script to insert all the indels (including the longer ones) from the .vcf into the .fasta. Last edited by kriikku; 01-13-2013 at 06:46 AM.

11-13-2011, 01:01 PM	#2
maubp Peter (Biopython etc) Location: Dundee, Scotland, UK Join Date: Jul 2009 Posts: 1,543	Are you trying to get the consensus sequence from a BAM file? The reference sequence would be the same standard human genome used for all the 1000 genomes, and can probably be downloaded from their site as FASTA.

11-13-2011, 02:50 PM	#3
mez Junior Member Location: georgia Join Date: Nov 2011 Posts: 5	Yes, I want to get the consensus FASTA sequence from the BAM file for a given chromosome region(not necessarily the whole chromosome sequence).

11-14-2011, 04:34 AM	#4
maubp Peter (Biopython etc) Location: Dundee, Scotland, UK Join Date: Jul 2009 Posts: 1,543	Have you tried searching the forum (or Google) for the terms BAM consensus? Look at the samtools mpileup command (which replaced the older samtools pileup command).

11-14-2011, 02:12 PM	#6
maubp Peter (Biopython etc) Location: Dundee, Scotland, UK Join Date: Jul 2009 Posts: 1,543	You're not the only person to hit that, http://biostar.stackexchange.com/que...ric-in-numeric