Questions tagged [samtools]

Questions specific to interacting with and post-processing sequence alignments using the SAMtools package. For questions specifically about formats SAM, BAM or CRAM use tags sam or bam,

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Interpreting 'samtools mpileup' output for multiple inputs

I would like to calculate sequencing coverage for a WGS project. Both long and short reads. I've used samtools as following: ...
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Detect mutation context in a read of a sam file

After sequencing (Illumina) of some DNA, I generated a sam file through alignment of a fastq file (using Bowtie2). Instead of using variant calling programs, I want to know the specific variances, ...
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Potential side effects of replacing read group tags in BAM file

I have a set of BAM files where the read group tags have some (default?) values, i.e.: @RG ID:RG0 LB:LB0 PU:PU0 SM:SM0 This creates issues in my downstream ...
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samtools view command not found error

When I tried to use samtools to split a bam file based on different chromosomes, I used this command: samtools view input.bam -b chr21 | chr21.bam However, I get ...
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BWA mem | samtools view: Intermittent parsing error

Update The issue was that bwa was running out of memory and failing, but that error wasn't floating to the top (see @Steve's answer, below). I was getting an error ...
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Use Htslib to create auxilary tags in bam file C++

I am creating a threaded c++ file where i generate in silico bam files, using header, DNA sequence and read information. First i use bam_init1() to create the bam1_t structure just named "b"....
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Samtools sort: most efficient approach to sort a single sample after aligning split .fastq files

Related to my other question (Samtools sort: most efficient memory and thread settings for many samples on a cluster), we need to optimize samtools sort as we ...
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Samtools sort: most efficient memory and thread settings for many samples on a cluster

We're preparing to analyze thousands of .bam files beginning with re-alignment, sorting, etc. Complicated question: Has anyone investigated the optimal thread and ...
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How to Sort and Index a SAM file without converting it to BAM?

Generally, I use samtools to sort and index BAM files. Samtools works also with BGZF ...
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How to analyze an IGV alignment

I'm working on a project where I am analyzing the performance of an alignment workflow. My goal is to find regions in the resulting BAM file where there are outstanding discrepancies or anything that ...
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1 answer
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Is there a sam flag for all&none of the reads?

a. Is there a SAM flag that specifies all reads? b. Is there a SAM flag that specifies none of the reads? So that if I run samtools view -f (a) -F (b) the result will be all reads of the file (as if ...
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Convert VCF file to mpileup.txt

I am working on an iterative analysis that uses orthologous pipelines that require mpileup.txt files as input for a visualization step. This requires me to convert VCF files to mpileup.txt. This ...
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How to extract all sequences mapped to a transcript from Kallisto output

This question has also been asked on Biostars I ran Kallisto with the --pseudobam option. How do I extract all the short reads that are mapped to a single transcript (e.g. ENST00000367969.8)? As a ...
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How to convert multiple single-end bam files to fastq using samtools

Hi I am trying to convert bam files generated from Ion Torrent Proton sequencing to fastq format so that I can upload them to ...
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2 votes
1 answer
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Counting the co-occurrence of variants A and B in an aligned sequencing read

I need some help getting started on this project. To simplify we want to be able to quantify the occurrence of 3 variants on each sequencing read in an alignment file as a proxy measurement for ...
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Is it normal to have a smaller bam file after merging bam files?

I have 2 bam files that belong to the same sample and I merged them with samtools merge. And after merging, I realized that the merged version is a bit smaller than ...
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1 vote
2 answers
118 views

Can HTSlib extract bam reads occurring in a specific region without iterating through the whole file?

I am using htslib/sam.h to write a C++ program. As part of this program, I must extract reads occurring on specific scaffolds in specific regions from a bam file. Essentially, I want to perform the ...
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3 votes
3 answers
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Unable to open .bam file in C++ using SeqAn due to 'seqan::UnknownExtensionError'

I am trying to open .bam files in C++ to extract reads occurring at specific scaffolds and loci. I essentially want to call "samtools view sample.bam -o sample.sam scaffold:pos-pos" from C++....
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Conversion of SAM to BAM files

I am very new to micro RNA analysis. I have been using H. sapiens, GRCh38 + major index as given in the Bowtie Website to align with my trimmed FASTQ file . The command I am using to make very ...
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2 votes
1 answer
38 views

Python package or CLI tool outputting mutations of sequence with respect to reference

I'm new to bioinformatics, and I'm looking for a tool that takes two FASTA files, one containing a reference, let's call it A.fasta, the other another sequence, let'...
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seqff script got trouble after samtools treatment

I'm trying to study fetal fraction by using a seqff script from here. The instruction says that I need a headless sam file to work so after alignment with bwa, I process my sam file by using samtools: ...
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3 votes
1 answer
404 views

Inflate operation failed: progress temporarily not possible, or in() / out() returned an error

I download a BAM file from ftp-trace.ncbi.nlm.nih.gov:/giab/ftp/data/NA12878/10Xgenomics_ChromiumGenome_LongRanger2.0_06202016/NA12878_GRCh38.bam (171G), and I am trying to view the comparison result ...
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How can I run the latest version of samtools on Slurm?

I am trying to run the following script on slurm to extract information from .sam files. While everything is perfect on my local machine with the samtools version 1.11, it does not work on slurm. I ...
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1 answer
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How to perform bowtie2 analysis with slurm?

I am trying to run my alignment script that works locally, using SBATCH. Official manual for bowtie2 says I can use ...
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1 vote
1 answer
371 views

How can I extract information from .sam files?

I have 10 .sam files after my bowtie2 alignment on ten single-pair sequences. I would like to build a graph based on that output ...
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1 vote
1 answer
644 views

What is "unmapped read segments" in the output of samtools idxstats?

samtools idxstats produces a four column output (see here) ...
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1 answer
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How to extract reads with INDELs > a given size?

I'm trying to modify this https://www.biostars.org/p/253774/ To get reads with deletions > 20bp I think this gives reads with exactly 20bp dels: ...
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2 votes
1 answer
901 views

How to generate a consensus sequence from a multi-reference BAM file?

I am trying to generate a consensus sequence from a BAM file that was generated by mapping reads to a reference FASTA containing multiple sequences. Usually, I generate consensus sequences from BAM ...
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1 answer
152 views

Reading fasta sequence regions with faidx

Im coding in c++ and reading in different reference genomes to examine regions across the chromosomes a few hundred basepairs at a time. To do it in c++ i use the library htslib and the command ...
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1 answer
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How to identify to each scaffold a read belongs to, inside a .sam file?

I have a fasta file assembly and combining it with the raw reads we produced a .bam file which I converted to .sam . The .sam information lines look like this: ...
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1 vote
2 answers
363 views

Trying to create a .bam file without the need for a .sam file

I'm trying to use the code specified in this link to create a .bam file without the need for a .sam file. Here is the code I'm using: ...
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Mapping statistics from bam file using bbtools and sambamba

Below are the statistics for RNA-seq mapped and unmapped paired-end reads to rice genome using reformat.sh from bbtools on bam files. It gives 77% mapped and 5% unmapped, what about the remaining 18% ...
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summarising read group information from a .bam file

I have merged together 2 different .bam files in order to simulate sample contamination. So the reads can come from one of two samples, as shown by the read group info: ...
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NO_COOR reads not in a single block at the end 0 -1

I am doing an RNA seq analysis with mouse samples. I had my reads in several lanes, so I concatenated those fastq reads. Then I've mapped my reads with Hisat2 against the new version of mouse "...
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2 answers
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Basic question about finding mRNA sequence in transcriptome

I am a computer scientist just starting out in bioinformatics topics, and would appreciate any guidance that can be given here: I have an mRNA sequence- an isoform - whose length is about 4000 base ...
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1 vote
1 answer
3k views

Samtools Index: Chromosome Blocks not Continuous

I am working with short-read whole-genome sequences from the NCBI's SRA. I have aligned and sorted all of my short-read sequences and am attempting to index each sequence into .bai format using ...
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2 votes
1 answer
342 views

How to extract unmatched reads using bwa and samtools?

I have a single read (NOT paired) that I need to pass through the workflow described in Beauclair et al. paper (free version here https://rnajournal.cshlp.org/content/24/10/1285.long) for identifying ...
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1 vote
1 answer
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How can I get unmatched reads for defective genomes analysis using bwa and samtools?

I am trying to follow the Beauclair et al. paper (free version here https://rnajournal.cshlp.org/content/24/10/1285.long) for identifying defective genomes using their DI-tector program. According to ...
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2 votes
4 answers
216 views

How to make work programs from the $PATH?

I am trying to analyze my RNAseq reads for defective genomes and I use this program (http://www.di-tector.cyame.eu/) that is suggested by Beauclair et al (https://pubmed.ncbi.nlm.nih.gov/30012569/) ...
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4 votes
1 answer
183 views

Is there a tool that can perform a read-group-aware mpileup from a single file?

I would like to perform a samtools mpileup from a single file that contains thousands of read groups with different SM tags. I could split the bam by read group ...
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2 votes
1 answer
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Phasing problem using "samtools phase"

so I'm having a problem using samtools phase. I'm trying to phase my .bam archives that where generated by aligning my samples with my reference genome. Following the documentation I tried the command:...
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2 votes
3 answers
384 views

Accessing .bam/.cram files from AWS S3?

What's the best/easiest way for accessing .bam/.cram files from S3? I have .bam/.bai .cram/.crai and .bed + .gff files that I want to make available easily to others so they can browse in IGV and ...
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1 vote
2 answers
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merging two/or more bed file into one bed file

I am trying to merge two bed files (more in future) to one. my bed files are something like : . I need to merge them in a way to have the shared chromosome location. Is there a way to do that ?
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2 votes
1 answer
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Cuffmerge: EOF marker is absent. The input is probably truncated

I need to merge my all transcripts.gtf from cufflinks output follow this command line : cuffmerge -o merged_gtf_output -p 15 -s ref.fasta -g anot.gtf assembly.txt ...
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1 vote
2 answers
759 views

Marking optical or PCR duplicates with picard vs. samtools flagstat

I am trying to identify the best library prep method for noninvasive prenatal test samples, to be sequenced with Illumina Novaseq. One metric that I am evaluating is the number/ percentage of PCR or ...
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1 vote
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Indel quality from mpileup

Could someone please confirm that I understand this correctly? I have the res and qual columns from mpileup and I would like to match them to get the qual per base. It seems that the indel initiation (...
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2 votes
1 answer
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Why does samtools mpileup sometimes include ref bases (other than ',' or '.')?

This is my first post here. I can think of no way of giving an easily reproducible example, as per the stack ethos. SO apologies in advance and any feedback on question format appreciated. I have ...
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1 vote
1 answer
788 views

How to filter a SAM file by a bed file?

I have a large BAM file and I want to filter it by just what's defined in a bed file, and write it to a new file. Is it possible to do this with samtools, and if so, how? Is there a better tool I ...
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1 vote
1 answer
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samtools / bamUtil | Meaning of <wildcard> as Reference Name

I have been working with several tools on bam files recently and I am not sure how I should interpret some of the outputs. samtools idxstats mapped.sorted.bam <...
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1 vote
1 answer
179 views

How to obtain file offsets corresponding to range query in block-gzipped file

How can we retrieve the file offsets of the zipped-chunks in a block-gzipped file that contain records for a region/window of interest (e.g. Chr01:100-Chr02:145)? It seems like the CLI of ...
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