Questions tagged [sam]
Use this tag for questions specifically related to the text SAM format. For general questions about the SAM/BAM formats use the tag BAM.
234
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29
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Discrepancy in Read Counts Between FastQ and BAM Files in Adapter-Trimmed Pipeline
In a FastQ to BAM pipeline where only adapter trimming is performed, I've noticed a potential discrepancy in read counts between the initial FastQ files and their resulting BAM file. Specifically, I'm ...
- 131
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35
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Generate simulated bam for certain snps
I want to benchmark my DNA sequencing pipeline. In order to do that I want some gold-standard files in every step(.vcf,.bam,.fastq). I want to generate/simulate a bam file of reads for a given set of ...
- 295
3
votes
1
answer
121
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low mapping percentage in bwa mem
After aligning paired-end reads to a reference genome, I am getting low percentage:
...
- 103
1
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1
answer
56
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obtaining a fragment data file bed from bam
Im trying to follow this pipeline https://github.com/epifluidlab/cragr on cfDNA WGS. I have bam files from paired end reads aligned with bwa mem.
The workflow requires bgzipped and indexed fragment ...
- 21
2
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1
answer
67
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How to subsample BAM based on read line number?
Consider a sorted, indexed, only mapped reads containing BAM file.
Is there a way to get a sub BAM based on read line numbers?
For example get a sub BAM file which contains read line numbers [1, 3, 8, ...
- 131
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47
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STACKS ref_map.pl no BAM records
I have 133 aligned and sorted BAM files that I am trying to run through the ref_map.pl script from the STACKS pipeline. My script looks as follows:
...
- 31
1
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1
answer
52
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Check mutation of a gene
Hi all, I am checking if a gene which around 50k base pairs has mutation. So is that a variant from A to G? Would you please tell me how to do so? Thank you so much!
The reference sequence from GRCh38,...
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-1
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1
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84
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Gviz Coverage Plots
This question has also been asked on Biostars
I have two bam files from single-cell RNA sequencing mapped to the reference genome using CellRanger, I can view them in IGV and I have a particular ...
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2
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23
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Off-target % for whole-exome sequencing panel
My samples have been sequenced with the Twist Exome 2.0 sequencing panel, and I want to assess how efficient the sequencing has been. By efficient, I mean examining metrics such as the uniformity (...
- 181
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27
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How to export the list of soft-clipped regions from a region of interest in IGV
I want to export the list of soft-clipped regions into a csv file from a region of interest in IGV. Additional information regarding the soft-clipped sequence, coordinate and nucleotide length will be ...
- 1
1
vote
1
answer
55
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Can bwameth accept unaligned Bam?
Does bwameth accept bam (unaligned) type as input or I will have to convert unaligned bam to fastq (samtools fastq bam) to use ...
- 645
2
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1
answer
162
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calculate mismatch frequency/rate from a BAM file
I am working with PAR-CLIP data where experimental procedure induces T to C mismatches. The other types of mismatches we can consider coming from artifact such as PCR errors. It could also be an ...
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14
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5
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2k
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Good / recommended way to archive fastq and bam files?
We have a lot of Illumina sequenced exome data. Currently we are using spring for its great lossless compression, but we are looking if there is anything better (and most preferably opensource) which ...
- 321
2
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1
answer
94
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Dealing With Manta Limitations
I am trying to figure out a way to remove the limiting factors that Manta says it cannot handle. To quote from the Manta page:
The following limitations exist on the input BAM or CRAM files provided ...
- 380
1
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1
answer
331
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Estimate insert size for single-end data with picard CollectInsertSizeMetrics
I have a BAM file generated from single-end data and I want to estimate the insert size using picard's CollectInsertSizeMetrics as follows:
...
- 335
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83
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Help sought with GWAS and vcf files, lack phenotype labels
This question has also been asked on Biostars
Hi, I am very new to this area, and I am taking a class about bioinformatics. For an independent project assignment, I need to do a GWAS. I am using the ...
1
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1
answer
210
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In CellRanger output, what are the reads with an xf:i:17 tag?
When running CellRanger on 10x scRNA-Seq data, a BAM file is output with the aligned reads. These reads have an xf tag, described here:
The bits of this tag are ...
- 374
3
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2
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196
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What can be the bias of aligning paired-reads in a single-end mode?
I don't know if I'm in the right place but I have a technical problem to fix. I would have to align paired-end reads from Illumina sequencing to compare a normal genome with a tumor one.
When I align ...
- 43
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1
answer
227
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Make mpileup file with several BAM files
I'm new to this business, so my question may seem as dumb. I want to make an mpileup file using a reference sequence and several (two to begin with) aligned sorted BAM files. And I want the output ...
- 3
2
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1
answer
97
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need bam file for pilon
I just ran an assembly on yeast genomes using Flye and I want to polish those assemblies with Pilon but it requires a sorted BAM file.
How do I make a BAM file of the resulting assembled.fasta?
- 923
2
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1
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147
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Count reads at specific gene features
I have BAM files from an RNA-Seq experiment and for all genes (or a subset) I want to get the number of reads in regions around the TSS (e.g. 2kbp) and the TES (e.g. 2 kbp) and calculate the ratio ...
- 335
2
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1
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669
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how to retain reads with low mapping quality (MAPQ) scores when using samtools view -q
I have been using the -q option of samtools view to filter out reads whose mapping quality (MAPQ) scores are below a given ...
1
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1
answer
150
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Aligning scRNA-seq fastq to .bam without cell barcodes
I would like to convert the fastq files from this dataset: https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE98679 to .bam files. The group did not provide the scripts they used to to their ...
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1
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147
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Get number of reads with a single, (almost) exact match to the full length of a reference sequence
I can't find an answer to this in previous questions, so hoping someone can help me now.
We have nanopore sequenced a PCR product, and I've filtered our reads to +/- 10 bp of the expected product ...
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6
votes
4
answers
761
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multi-sequence alignment of samples with multiple contigs each
I want to perform a multi-sequence alignment on 12 samples that clustered based on cgMLST. Ultimate goal is to find out whether they differ by the presence of a few genes.
I have performed multi-...
- 283
2
votes
1
answer
173
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Filtering paired-end reads with sambamba: avoid discarding reads on the minus strand
I have a BAM file (DNA, shallow whole genome sequencing at ~1X) where I want to filter reads (using sambamba) to keep only those which have a template length > 20 and mapping quality > 20, ...
- 131
2
votes
1
answer
312
views
How does split reads look like in sam files?
I used bwa mem to align the DNA with the reference genome. If there are split reads (chimeric reads, come from two different parts of the genome), will it be split into two lines rather than one line?
- 101
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297
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Can I use samtools addreplacerg to replace multiple RG entries at the same time?
I have a bam file that contains two @RG lines:
...
- 113
1
vote
2
answers
119
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Filter bam with sambamba
I have a very large bam file and I want to filter it to keep only a handfull of positions as defined in a bed file. Can I do this with sambamba or do i need another tool?
Thanks
- 121
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2
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49
views
BAM files with no RNAME and POS, how to map contents to SNPs?
I have a set of 4 .bam files containing the exome of an individual, around 400 MB each. I used samtools to generate a 2.4 GB .sam file out of one of the .bam files, and I found it contains lines with ...
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347
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How to extract reads that map exclusively to a single site with 1 or zero mismatches from BAM files
I generated BAM files (sorted by coordinates) by aligning human RNA reads against the human reference genome using BWA MEM and ...
- 21
1
vote
1
answer
313
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VCF or BAM file for raw data of gene test?
My friend has a VUS (Polymicrogyria with or without VEDS) mutation that was found in her whole-exome sequencing with respect to the phenotype given that time. At present, her doctors have more ...
1
vote
1
answer
65
views
Reference variant detected as altered one in bam file
I received (from manufacturer) several .bam files and I used four callers (samtools, freebayes, haplotypecaller, deepvariant) to find some sequence variants. In obtained .vcf files, I took a closer ...
- 206
3
votes
2
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148
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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, ...
- 33
1
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2
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810
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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 ...
- 1,783
4
votes
1
answer
351
views
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 ...
- 135
2
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2
answers
4k
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How to convert a BAM file into a VCF file
I know that there is a lot of methods to do so, but most of them need to have a reference file. (For example, the GATK force me to provide a reference.)
However, I think the BAM file I am working with ...
- 135
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3k
views
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 ...
- 103
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0
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58
views
Under what circumstances can the SEQ field in a SAM file be a *?
I saw in the SAM format specifications that the SEQ field (10th column) can be a "*" if the sequence is not stored, instead of being the sequence of the mapped read. Under what circumstances ...
- 21
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0
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425
views
pysam "Exec format" error
I am a beginner and trying to read a bam file in Python.
The lines below throw the error
...
- 11
1
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1
answer
56
views
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 ...
- 279
6
votes
1
answer
623
views
Why do SAM and BAM use different coordinate systems?
BAM files are, at least as far as I know, simply binary compressed versions of SAM files. They have the exact same information and are used in the same way. Why then does the SAM format use a 1-based ...
- 9,662
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33
views
Fastest way to count where two bam files are homozygote reference (inverted variant calling)
Maybe I'm having a brain-freeze so excuse me if this is a waste of everyone's time...
I am working with an organism with ~700Mb genome.
I have bam files of two individuals that are whole genome ...
- 121
1
vote
2
answers
437
views
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 ...
- 21
1
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263
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Extract read names and the associated nucleotides on specific positions from a BAM file (in R)
Let's assume I have a BAM file and several positions that I would like to examine more closely in this alignment. My goal is to find out whether these positions are on the same reads and which ...
- 11
1
vote
1
answer
584
views
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 ...
- 41
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votes
0
answers
150
views
Splitting .bam files into separate samples for tophat2
I have RNA seq data of 12 samples (paired end, so 24 files) and I ran tophat2 for alignment on all of them which gave me one bam file for all the samples. Now I need separate bam files for all the ...
2
votes
1
answer
52
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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 ...
- 21
0
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1
answer
486
views
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 ...
1
vote
2
answers
348
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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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