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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38 views

How do you map nanopore fast5 files?

Is there a best practices for mapping Oxford Nanopore files to a reference? Is there a tool that can take a tarball of fast5 files and map them directly or do they need to be converted to fastq first?...
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How to choose reference to calculate coverage of WGS of organism that has high intra-species variation?

Calculation of coverage involves mapping reads with a reference and depending on the choice of reference we can get very different result. If a species has a genome that has very low size variation ...
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Error with BWA Mem input having multiple fastq files using cat and process substitution

Running BWA Mem on a number of paired end fastq files using process substitution on the inputs results in this error: ...
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1answer
43 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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41 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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75 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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70 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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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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258 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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46 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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69 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 ...
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92 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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91 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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62 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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621 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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200 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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63 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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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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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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77 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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691 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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372 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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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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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 ...
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1answer
122 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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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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979 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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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.: ...
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85 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
98 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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456 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
79 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
697 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: ...
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1answer
2k 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 ...
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1answer
810 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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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
402 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 ...
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594 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)...
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653 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 ...
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134 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) ...
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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 ...
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2answers
506 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 ...
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1answer
61 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 ...
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1answer
989 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 ...
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4answers
189 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 ...
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198 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 ...
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179 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 ...
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1answer
88 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 ...
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2answers
190 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 ...