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Questions tagged [read-mapping]

When dealing with associating sequencing reads to a matching position in a set of reference sequences (typically a genome).

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1answer
17 views

What do the symbols mean in minimap2's gap cost equation?

Heng Li's paper on minimap2 is understandable to me right up until the moment that it gives this definition of gap cost: 2.2.1 Alignment with 2-piece affine gap cost Minimap2 performs DP-based ...
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38 views

How to obtain the extended unaligned bases of the read in reference based genome assembly?

I did illumina read mapping based on a particular gene sequence (reference sequence) using the following command, ...
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1answer
58 views

Oxford Nanopore mapping Quality and sequencing error

I'm analyzing some Oxford Nanopore sequencing reads, with several articles stating the sequencing error is between 5% and 15%. Since its 1D and not 1D^2 sequencing i would assume the sequencing error ...
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2answers
49 views

How can I transform a mapped BAM file into an unmapped BAM file?

In order to use MergeBamAlignment (Picard), I need an unmapped BAM file and a mapped BAM file. I have two mapped BAM files: one with reads mapped to the reference I want but without metadata such as ...
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0answers
21 views

RNASeq read coverage in protein space?

I used samtools depth to find the per base-pair read coverage over a number of isoform contigs from my Trinity assembly. I have also conducted a multiple sequence ...
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1answer
111 views

bowtie2 options when mapping stranded single end reads

I'm trying to map RNA-Seq reads generated using the NEB Ultra Directional kit is first strand reversed protocal. I'm using bowtie-2.3.5_1 to align against the mus ...
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1answer
44 views

No refseq transcripts in 2nd half of Y chromosome for HG38

Obviously there is a gap in my understanding of how sex chromosomes are annotated. I've been working on some CNV calls and noticed that most of the 2nd half of ChrY is missing annotations and no reads ...
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58 views

Merging/concatenating index files

I was wondering if any one has had success merging pre-generated reference index files for BWA, bowtie2 from multiple references? For a concrete example I have hg19 and a bunch of viral and ...
4
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1answer
66 views

Displaying soft-clipped nucleotides in samtools tview

There are nicer genomics visualization tools available, but the samtools tview command is almost always my go-to for a quick first look at read alignments. I just ...
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1answer
50 views

Batch alignment of inconsistently named Fastq files

I have numerous gzip-compressed paired-end Fastq files with ChIP-seq data that I would like to align with bowtie2. I confirmed that bowtie2 takes .gz files, ...
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0answers
47 views

How snippy makes MSA-like aligned fasta output from multiple samples?

From the log file it seems snippy doesn't do assembly. It only does mapping: fastq --> SAM --> BAM --> VCF --> consensus_seq/snps But if multiple ...
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2answers
279 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?
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1answer
108 views

HISAT2 - are all multi mapped reads reported by default?

While going through the HISAT2 manual, one can see this: Two alignments for the same individual read are "distinct" if they map the same read to different places. Specifically, we say that two ...
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2answers
47 views

bedtools: get name column of alignment file A

I'm using the coverage function of bedtools to check whether a certain set of regions (file B) has any overlap with known ...
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32 views

What Ensembl genome version of wheat should I use for alignments? [duplicate]

There are many genome files available from Ensembl. Which one is the best to use/download for wheat RNA seq differential gene expression analysis? Also, tell me which tool or aligner or mapper to use? ...
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1answer
74 views

Highly heterozygous reads mapping

I have short (67bp) Hi-C reads from a highly heterozygous organism (~15% between-haplotype SNP divergence) and I have both reference haplotypes. I wanted to try and compare different haplotyping ...
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2answers
64 views

Control width of samtools tview “snapshot” when redirecting

I have a large number of loci I would like to examine manually with samtools tview. Rather than typing or copy-n-pasting dozens of coordinates, I was hoping to ...
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2answers
456 views

How to count the number of mapped read in 100-bp window from a BAM/SAM file

Although I know how to get total number of mapped read using samtools flagstat (samtools flagstat file_sorted.bam) but I want to count total number of mapped read ...
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2answers
280 views

What is mate rescue in bwa mem?

BWA mem has the -S and -P tags for skip mate rescue and skip pairing; mate rescue performed unless -S also in use. What do ...
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0answers
37 views

Calculating alignment/mapping time

I am trying to assemble a plant genome using AWS resources using velvet. Plant genome is huge (> 10 times human genome) and coverage is around 30 x. We are planning for de novo assembly with Velvet (...
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3answers
160 views

Is there a read mapper that allows input in SAM format instead of FASTA/FASTQ?

When we get raw sequenced reads in FASTQ format, we usually need to do some pre-processing (QC, demultiplexing, etc.) prior to sequence alignment. The only way to register the results of this ...
3
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1answer
90 views

Problem: “Pair-end” reads scRNA seq data (Drop-seq)

In case of Drop-seq, we have paired end data. Read 1: Cell code + UMI (unique molecule identifier) Read 2: The transcript information But I have a problem/doubt with the sample I am working on. ...
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0answers
19 views

Visual representation of detecting deletion by different read types

I'm having some trouble differentiating between CNV, split reads and read pairs and how each one elucidates the presence of a structural variation, say deletion. Given a reference and donor sequence,...
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1answer
553 views

What are “split reads” and “intron clusters?”

I'm working through the example data set for LeafCutter and the documentation mentions "split reads": This will cluster together the introns fond [sic] in the junc files listed in test_juncfiles....
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1answer
30 views

Does read sequence identifier need to be unique for proper alignment?

I am preprocessing a set of scTHSSeq reads in which each read sequence identifier has been replaced with the cell barcode, e.g.: ...
4
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1answer
77 views

Reference genome for allele specific expression

We are trying to sort out a pipeline for doing allele specific expression. Our plan is to call SNPs from RNA-seq data and combine with known SNP annotations. A well known problem in ASE is reference ...
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3answers
81 views

How to detect mismatch before mapping in RNA-Seq data

In the methods of this paper, the authors say: Reads were then filtered based on quality score in the UMI region. Any read with >1 low-quality base (phred <=10) were discarded. Reads with more ...
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2answers
317 views

What is the correct way to map Hi-C data with bwa mem?

Library Prep I have a Hi-C library prepped using an enzyme that cuts at GATC, so it leaves GATCGATC as the junction sequences. ...
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2answers
70 views

How to map reads shorter than 32bp with minimap2?

I mapped fasta sequences to a reference. The fasta sequences range in length from 17bp up to several hundred bp. I used minimap2 with the following command: ...
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1answer
510 views

Can I run STAR without an annotation file?

I wish to use Rascaf to scaffold a fragmented draft genome. For this, I need to provide a BAM file of aligned RNA-seq reads and the draft genome. So, I indexed the draft genome with STAR like this: ...
3
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1answer
1k views

Hisat2 : which option should mention for strand specific library read

I was trying hisat2 I get confused by the strand options... The question has been already asked on github here but didn't get any satisfactory answer. I'm surprise how much this strand information ...
3
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1answer
635 views

Strange per sequence GC content results

I would be grateful if someone could take a quick look at these FASTQC results. This is rna-seq paired-end data. From the FASTQC manual, an unusual distribution seems to be suggestive of contamination ...
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2answers
40 views

Mapping bisulfite reads to a reduced size genome

Is it worth mapping bisulfite reads (WGBS) to a reduced size genome? I'm interested only in modifications in CpG context, thus instead of mapping to a whole genome (human genome) I would: Extract ...
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2answers
354 views

Mapping Information from SAM/BAM file

I mapped raw illumina reads to longer pacbio reads and I would like to know the following information from my mapping file (SAM/BAM) How many PacBio reads are mapped to at least one illumina reads A ...
6
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1answer
497 views

How to trim adapter sequences from GSE65360 in order to map the reads?

I am trying to map single-cell chromatin accessibility data from doi:10.1038/nature14590, obtained using scATAC-seq, to the reference genome. Example paired-end reads are here. The reads contain ...
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4answers
2k views

Why total RNA-seq usually yields low mapping rate?

Maybe this is a silly question, but I'm really wondering why we usually get low mapping rates if we map total RNA-seq, but not poly(A)-enriched (in particular, for human, mouse, and zebrafish datasets)...
5
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1answer
529 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
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2answers
128 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) ...
11
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2answers
4k 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 ...
4
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2answers
434 views

Problems to Extract uniquely mapping reads from BWA MEM alignment

I did a mapping of genomic paired-end reads to a reference assembly using bwa mem. I need to extract the reads that mapped only once to my reference. For that, I have tried to follow the method ...
4
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1answer
60 views

Best way to detect long insertions in bisulfite sequencing data?

I am interested in identifying indels in whole genome bisulfite sequencing data (76bp paired end). Currently, I do this by setting the -rfg and ...
3
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1answer
830 views

Counts obtained by featureCounts seem much less than observed coverage

I have surprisingly low counts when running featureCounts on some (single-end) RNA-seq data mapped on C. elegans genome using ...
1
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4answers
167 views

At what processing step should library strandedness type be taken into account?

Suppose I have single-end RNA-seq data for which the reads in the fastq file are reversed with respect to the original extracted RNAs. Suppose I have the following workflow: Map the reads on the ...
4
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3answers
176 views

what percentage of the human genome is MAPQ=0?

When doing Illumina 2x150bp sequencing of genomic DNA, and after aligning the reads to GRCh38, what percentage of the non-N fraction of the human genome is MAPQ=0? This is, what part corresponds to ...
6
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3answers
160 views

Filter Trinity transcriptome based on RNASeq reads

I have recently generated a genome-guided transcriptome with Trinity, and would like to apply an additional filter to exclude transcripts that don't have good support from the RNASeq reads. This is ...
7
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1answer
83 views

How to interpret contig-alignment.psa produced by velvet

I'm using velvet to align given reads of RNA to given CDSs (i.e. coding areas and genes) of an organism, so I can generate gene-expression profiles. But after using ...
5
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2answers
185 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 ...
9
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3answers
296 views

Visualisation of long read RNA-Seq splicing

I have a dataset of Oxford Nanopore cDNA reads. Many of my reads are full-length or close to full-length transcripts, and I and am interested in examining alternative splicing. For this, I would like ...
5
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1answer
690 views

How do I generate a variant list (i.e. VCF file) using Illumina reads from a human genome?

This is a problem I have to solve frequently, and I'd be interested in knowing what other methods people use to solve the same problem. About twice a year, I get asked to determine variants from ...
11
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4answers
359 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. ...