Questions tagged [alignment]

These questions are about sequence alignment.

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16
votes
2answers
8k views

Obtaining uniquely mapped reads from BWA mem alignment

This is based on a question from betsy.s.collins on BioStars. The original post can be found here. Does anyone have any suggestions for other tags or filtering steps on BWA-generated BAM files that ...
15
votes
2answers
7k views

Merge hundreds of small BAM files into a single BAM file

I am working with over a million (long) reads, and aligning them to a large genome. I am considering running my alignment jobs in parallel, distributing horizontally across hundreds of nodes rather ...
14
votes
1answer
3k views

How does the BWA-MEM algorithm assign its mapping qualities?

Is there any resource (paper, blogpost, Github gist, etc.) describing the BWA-MEM algorithm for assigning mapping qualities? I vaguely remember that I have somewhere seen a formula for SE reads, which ...
13
votes
2answers
8k views

Meaning of BWA-MEM MAPQ scores

Does anyone know what the MAPQ values produced by BWA-MEM mean? I'm looking for something similar to what Keith Bradnam ...
13
votes
1answer
202 views

Compare alignment quality of multiple sequencing runs aligned against the same reference genome

I have run Oxford Nanopore Technologies' MinION sequencing on the same DNA sample using three flowcells, each aligned against the same reference genome (E.coli K12 MG1655) using both BWA MEM and ...
12
votes
4answers
507 views

Library for computing BWT-based alignments

I am writing a software tool to which I would like to add the ability to compute alignments using the efficient Burrows-Wheeler Transform (BWT) approach made popular by tools such as BWA and Bowtie. ...
12
votes
1answer
3k views

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

After some google searches, I found multiple tools with overlapping functionality for viewing, merging, pileuping, etc. I have not got time to try these tools, so will just see if anyone already know ...
11
votes
2answers
2k views

Difference between BWA-backtrack and BWA-MEM

Many of my colleagues recommend I use BWA-MEM instead of regular old BWA. The problem is I don't understand why and reading the BWA man page doesn't seem to help the matter. What is the difference ...
11
votes
1answer
4k views

Better aligner than bowtie2?

Bowtie2 is probably the most widely used aligner because of it's speed. Burrow-wheeler (BW) algorithms (including bwa) tend to ...
9
votes
1answer
165 views

Chimera Alignments

I have a structure with two subunits. I am trying to show movement of the C-terminal subunit upon ligand binding by superposition with another structure from the same strain in the apo form. I want ...
8
votes
2answers
528 views

Building STAR Genome Index for nanopore RNA sequencing

I am aligning a dataset of 1,000,000 reads oh human mRNA sequenced on Oxford Nanopore Technologies' MinION, and would like to use the STAR aligner, using the parameters recommended by Pacific ...
8
votes
2answers
993 views

Is there a standard definition for “assembly polishing”?

Is there a standard definition for "assembly polishing" in the field? Is there a standard definition for what polishing algorithms do? My understanding of "polishing" is strongly influenced by ...
8
votes
3answers
339 views

Books on bioinformatics algorithms

I'm looking for a book about bioinformatics algorithms, such as alignment, BLAST search, and variant calling. I'm hoping reading about this subject will give me a deeper understanding of the ...
8
votes
1answer
374 views

Aligning many long sequences

I'm faced with having to align many (some 100s) bacterial genomes, where the genome length is in the millions. Obviously, this is beyond normal alignment techniques and it's unclear to me what the ...
7
votes
3answers
2k views

visualisation of genome alignment

I was asked to check the synteny of some genes in two genome assemblies of the same species (PacBio & Illumina). I was given two scaffolds couples (so 4 scaffolds total) in the Illumina genome and ...
7
votes
1answer
556 views

The effects of incomplete bisulfite conversion upon mapping efficiency

This question has also been posted on Biostars I have sequenced numerous multiplexed pools of BS amplicon-seq libraries derived from human samples on a MiSeq over the past few weeks. I have been ...
7
votes
1answer
571 views

Interpreting Intergrative Genomic Viewer (IGV)

I was following a tutorial on "Tuxedo Genome Guided Transcriptome Assembly Workshop" and was wondering how to interpret the following: From what I understand from 'Color Legends', the color blue ...
7
votes
1answer
542 views

Extracting strand specific reads from MinION cDNA-PCR protocol

I recently performed my first MinION run, and now I'm trying to analyze the data. Being pretty new to the bioinformatics field, I was hoping some of you could help me out. As a bit of background, I'...
7
votes
2answers
496 views

Understanding the significance of BLAT score calculations for read fragment alignment

I'm attempting to reconstruct the read fragments that form a hit to a target, excluding/skipping gaps. BLAST's outfmt 6, and alternatives such as Diamond's ...
7
votes
1answer
416 views

Reject reads with low quality bases from a Bam file through pysam

I have a code below: ...
6
votes
2answers
2k views

Definition of “seed” in sequence alignment

I would like to know what is meant by "seed" for various sequence aligners. How is it important?
6
votes
3answers
421 views

tools to reconcile experimental transcripts with reference annotation

Looking for tools to reconcile alignment file of experimental transcripts mapped to genome (SAM/BAM) with the reference transcriptome annotation (GTF) from Ensembl (organism: D. melanogaster). The ...
6
votes
3answers
646 views

Total reads aligning to each reference within a bam file

I have two PCR amplicons that have been multiplexed and sequenced using the nanopore minion. I have aligned the fastq reads using minimap2 with a reference file containing both amplicon sequences and ...
6
votes
3answers
496 views

Generating the reconstructed alignment from BAM

I have a (small) BAM file with CIGAR and MD fields. Question 1: What tools exists in Python and/or R to reconstruct the alignment between the reference and the read in a BAM? Given that this is a ...
6
votes
2answers
283 views

Sequence alignment using Markov Model

I am learning about applying Markov model to sequence alignment. The prof says that the transition probabilities from a gap-residue alignment to a residue-gap alignment and vice versa are both 0. Is ...
6
votes
1answer
249 views

Y Chromosome Aligned Reads in scATAC-seq data from a female-derived cell line?

I'm working with scATAC-Seq data on the K562 cell line, which is supposed to be derived from a female patient. While following the scATAC-seq data analysis pipeline, after performing bowtie alignment ...
6
votes
1answer
104 views

Does the DNA or RNA of some ethnic groups map better than others' to the human reference genome sequence?

I believe that the human genome reference took DNA samples from different people and that some natural variation is included in extra contigs. However, the human reference genome comes from a ...
6
votes
2answers
238 views

How can I output an identity matrix in progressiveMauve?

I'm just getting started with the Mauve aligner and I'm finding the documentation a bit lacking. I'm using the progressiveMauve tool from the command line and would ...
5
votes
6answers
1k views

Identifying Indels from Chromatograms

I have around 100 chromatograms (.ab1 files) from Sanger sequencing a genome at loci believed to have an indel. I'm new to interpreting this kind of data in ...
5
votes
3answers
2k views

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

I am not talking about consensus sequence, I know how to get consensus sequence using mpileup in samtools/bcftools. As I understand , SAM/BAM files are basically sequence alignment format so it's ...
5
votes
2answers
205 views

Is there a Python/R package with the ability to convert an alignment and reference into a CIGAR?

I'm writing a python function from scratch to do this, but I feel like this must exist in some standard bioinformatics library already. In principle, this is a simply regex operation which many must ...
5
votes
2answers
128 views

Counting letters in phylip alignment columns with Biopython

I have been using python 3.6 and biopython 1.72 to work with protein data files. I am using a protein sequence file (phylip format), for example: ...
5
votes
2answers
617 views

How are Principal Component analyses and Admixture analyses from a genetic alignment different?

How are Principal Component analyses and Admixture analyses from a genetic alignment different? My understanding is that a PCA will take raw genetic differences across the entire alignment and plot ...
5
votes
1answer
1k views

What is local realignment and what is the problem it solves?

I am trying to understand local realignment but I could not get a clear idea of what is the problem solved by it. For example, reading Homer and Nelson (2010): Because alignment algorithms map ...
5
votes
3answers
185 views

Show presence of known mutation in RNA-seq data

We have RNA-seq fastq data from control (WT) patients and a patient with a point mutation at a known location in one gene. I'd like to retrieve the reads aligning to that gene and show the presence of ...
5
votes
2answers
177 views

RapMap: reference transcriptome for simulated reads

I am very new to bioinformatics and trying to repeat the benchmark in the RapMap paper with an experimental tool working in a similar but different fashion. In the paper (taken from their github) ...
5
votes
1answer
131 views

Why is a PacBio read length larger than the aligned reference region?

I recently had some Iso-Seq sequencing done on my organism catfish on the new Sequel platform and got weird alignments for a size selected 4 Kilobase and up fraction after running the isoseq3 pipeline....
5
votes
1answer
444 views

How to output all sequences with bwa mem, not `*`?

I've been running bwa mem -a for alignment, using the -a flag---this will output all alignments for SE or unpaired PE I've ...
5
votes
2answers
5k views

Difference between samtools mark duplicates and samtools remove duplicates?

What is difference between samtools mark duplicates and remove duplicates ? Is it necessary to mark duplicates before removing duplicates with samtools?
5
votes
2answers
695 views

Reads mapped to exonic, intronic and intergenic regions

After the alignment step I checked the rnaseq metrics of all the samples. Among 40 samples three samples show high percentage of reads mapped to intronic regions. What could be the reason? ...
5
votes
1answer
269 views

Why doesn't Biopython AlignIO.read() recognise the 'mauve' format?

On this Biopython tutorial, they describe how to import a multiple sequence alignment in the Mauve (XMFA: extensible multi fasta format). So I imported the AlignIO module: ...
5
votes
2answers
538 views

Is it possible to do alignment within Python? Check variants against reference?

I'm currently adding a few SNPs randomly into a FASTA within python using BioPython. In the following example from BioPython, I add an SNV at location "5" http://biopython.org/DIST/docs/api/Bio.Seq....
5
votes
2answers
194 views

How to estimate whether a long-read is meaningful sequence?

The setup Imagine that I work on an organism without a reference genome, and that the closest reference genome I can get is quite diverged. E.g. ~10% diverged in terms of SNVs when measured with ...
5
votes
1answer
548 views

Does the “.full.aln” file produced by snippy-core contain all bases of my input sequences aligned to the reference genome?

I have a number of sequences and a reference genome. I used snippy to align each individual sequence with the reference genome. I then used ...
5
votes
1answer
82 views

Are there certain alignment methods/tools which perform better with a high density of indels?

I have a set of experiments which should result in WGS reads with a high density of indels. Question: Are there certain alignment tools/methods which perform respectively "better" with these reads? ...
4
votes
2answers
276 views

How to align output of grep --color=always? (To QC fasta/fastq files)

Grepping out short sequences from a fasta or fastq file is a really useful way to look at sequencing data. Using the option --color=always makes this even more ...
4
votes
2answers
324 views

Reordering scaffolds according to a reference without a genetic map

I am trying to reorder scaffolds of a rice species, but no genetic map is available right now. Oryza sativa Japonica is a close relative of this rice species. Mummer was used to do a whole genome ...
4
votes
1answer
252 views

Can blat use more than one core/CPU to speed up the alignment?

I am using BLAT to align two versions of the genome of C. elegans. I can see in the Activity Monitor of my Mac Book Pro High Sierra that blat is using 100% of a CPU....
4
votes
2answers
981 views

How to align genomic sequence with corresponding amino acid sequence

Does anyone know of a program that can align a genomic sequence with introns with the corresponding amino acid sequence? I have both the genomic sequence and the correct amino acid sequence but no ...
4
votes
1answer
205 views

Efficiently aligning a lot of reads on the same small reference sequence

The context: I have a DNA-sequence coding for a protein, about 1500 bp in length. Using NGS, a lot of reads of (mutants of) this same sequence were acquired. All of these reads need to be aligned to ...