15 votes

What is the difference between samtools, bamtools, picard, sambamba and biobambam?

The obvious answer is that different people wrote them. It's fairly common in bioinformatics for people with a computer science background to get frustrated with existing tools and create their own ...
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  • 11.7k
15 votes
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Better aligner than bowtie2?

Bowtie2 is no longer the fastest aligner. Salmon and Kallisto are much faster, but have been designed to optimise RNASeq mapping. Their speed is gained from avoiding a strict base-to-base alignment, ...
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  • 11.7k
14 votes
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Merge hundreds of small BAM files into a single BAM file

samtools merge merged.bam *.bam is efficient enough since the input files are sorted. You can get a bit faster with sambamba and/or biobambam, but they're not ...
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  • 19.2k
14 votes
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Meaning of BWA-MEM MAPQ scores

First of all, if you want to understand mapping quality (mapQ), ignore RNA-seq mappers. They often produce misleading mapQ because mapQ is not important to RNA-seq anyway. Strictly speaking, you have ...
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  • 5,735
13 votes
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Obtaining uniquely mapped reads from BWA mem alignment

To exclude all possible multi-mapped reads from a BWA-mapped BAM file, it looks like you need to use grep on the uncompressed SAM fields: ...
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  • 11.7k
12 votes

Difference between BWA-backtrack and BWA-MEM

TL;DR: BWA-backtrack is based on backtracking. This approach is appropriate only when the dissimilarity between the reads and the reference is low, or when you want to find all best hits or enumerate ...
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12 votes
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Definition of "seed" in sequence alignment

The seed is the subset of a read used in the first step of an alignment. Many aligners work by a seed-and-extend model, wherein they first find all regions matching the "seed" and then extend the ...
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  • 19.2k
11 votes
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Library for computing BWT-based alignments

First, let us remark that there exist several hundred read mappers, most of which have been even published (see, e.g., pages 25-29 of this thesis). Developing a new mapper probably makes sense only as ...
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9 votes
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Aligning many long sequences

Whole genome aliment can be done using Progressive Mauve, LAST or Mummer. For bacteria I used Mauve since it has also very nice visualisation engine. A very new tool is Minimap2, a super fast mapper ...
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  • 5,287
9 votes

Library for computing BWT-based alignments

BWA-MEM can be used as a library. File bwa/example.c shows the basic functionality for single-end mapping. It should give identical mapping to the bwa-mem command line. Header bwa/bwamem.h contains ...
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  • 5,735
9 votes

Difference between BWA-backtrack and BWA-MEM

To quote the Introduction to BWA on sourceforge: BWA is a software package for mapping low-divergent sequences against a large reference genome, such as the ...
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9 votes
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How to count the number of mapped read in 100-bp window from a BAM/SAM file

one-liner Here's a gritty one-liner to count the number of reads in a region if you have just one region that you want to investigate. Change the 1 in ...
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  • 3,071
8 votes

Merge hundreds of small BAM files into a single BAM file

Merging sorted files is a linear operation, so any well-implemented tools that do it will do it with approximately the same efficiency. So samtools merge (use the ...
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8 votes
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Building STAR Genome Index for nanopore RNA sequencing

The parameter is used to determine how much sequence STAR indexes on each side of a splice junction to improve its alignment accuracy. For very long reads, this may not be ideal. I am not sure if ...
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  • 727
8 votes
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Total reads aligning to each reference within a bam file

The quick way to get the number of alignments on each reference is samtools idxstats my_bam.bam Number of reads on each reference is column 3. Although, as has ...
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  • 3,211
8 votes

How to get fasta alignment file from SAM/BAM file?

I am not sure what you mean by "fasta alignment file". If you mean a multi-sequence alignment (MSA) in the fasta format, you can't get that because SAM keeps pairwise alignments only and doesn't align ...
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  • 5,735
7 votes
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Y Chromosome Aligned Reads in scATAC-seq data from a female-derived cell line?

There are homologous regions between X an Y chromosomes: https://en.wikipedia.org/wiki/Pseudoautosomal_region It is therefore normal to have some female-derived reads mapping in Y chromosome. You ...
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  • 2,960
7 votes

The effects of incomplete bisulfite conversion upon mapping efficiency

Bisulfite conversion efficiency has no effect on the mapping rate in bismark and similar tools. The reason is that the reads are fully bisulfite converted in silico before alignment to minimize ...
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  • 19.2k
7 votes
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What is local realignment and what is the problem it solves?

SNPs are likely to be created and InDels are likely to be missed. Suppose you have a read, ACTGACTGACTGTAC and you align it to a reference sequence ...
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  • 19.2k
7 votes

why in RNA seq don't we only use reference transcriptome?

Pseudoaligmnent to a reference transcriptome with something like Kallisto is perfectly valid, assuming you are in an organism whose reference transcriptome is very well documented. But if you want to ...
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  • 1,662
6 votes
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Compare alignment quality of multiple sequencing runs aligned against the same reference genome

Qualimap will do this for you. Go to qualimap.bioinfo.cipf.es Run qualimap (default params are fine) on each BAM file Open up the HTML output, and you can read off the %identity (they measure the ...
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  • 952
6 votes
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tools to reconcile experimental transcripts with reference annotation

I've never tried this myself, so I don't know how easy this is... One option would be to start with GMAP, which is meant to align whole transcripts against the genome. The really nice thing about ...
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  • 19.2k
6 votes

Interpreting Intergrative Genomic Viewer (IGV)

You've apparently colored your alignments by read strand. In this case, red indicates "+ (watson) strand" and blue indicates "- (strand) strand". This strand association is determined by the ...
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  • 19.2k
6 votes
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Does the DNA or RNA of some ethnic groups map better than others' to the human reference genome sequence?

For simple variants like SNPs it would not really be a problem to use the current genome assembly for other ethnic groups. But for more complex variants this could be indeed problematic, however not ...
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  • 3,541
6 votes
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Is there a standard definition for "assembly polishing"?

In long-read assembly, "polish" refers to the step to improve the base accuracy of contig sequences. I believe the terminology was originated from the HGAP paper: The final consensus–calling ...
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  • 5,735
6 votes
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Sequence alignment using Markov Model

If I understand your question correctly, then I think for case of pairwise alignment, there is a simple explanation. I believe the key insight is that: a mismatch should always score better than a ...
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  • 3,462
6 votes
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How to align output of grep --color=always? (To QC fasta/fastq files)

Perhaps, grep is not the best tool to use in this case, but it should be in principle possible by using grep & ...
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6 votes
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Can blat use more than one core/CPU to speed up the alignment?

BLAT can only use one CPU. It is actually not the right tool for full-genome alignment. For "two versions" of the same species, MUMmer and minimap2 are orders of magnitude faster and probably give ...
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  • 5,735
6 votes

Definition of "seed" in sequence alignment

Devon's answer gives a good, concise definition. But it's also helpful to consider why seed-and-extend is used and what benefits it provides. Finding approximate string matches requires operations ...
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6 votes

Frequency of specific viral sequence in .BAM or .fastq

Most read aligners will report unaligned reads as well, which presumably will include your viral sequences. I would ask them to formally confirm that the BAM files will contain unaligned reads before ...
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