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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37 views

using Bowtie to map miRNA-seq data to rfam and also reference genome

I am using Bowtie to remove non-coding RNAs (tRNA, snRNA, rRNA) by rfam and also maping our microRNA-seq data with mirbase using following code: ...
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49 views

Solutions to reference bias issue

I've recently learned about the reference bias issue - inability to properly map NGS reads in certain genomic regions caused by the fact that our standard genomic references are linear and do not ...
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73 views

What is the meaning of split read?

I want to use rna seq data to later perform functional tests on fusion genes. so before that I need to filter the "best results" (of rnaseq) for deciding which candidates I actually want to ...
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20 views

Modeling number of reads mapped to a gene

I am looking for a probability distribution of a number of reads mapped to a particular gene in metagenomic sequencing (NGS, shotgun, likely illumina). Naively one could model it via a binomial (or ...
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67 views

How to check what software has been used to generate bam file?

I have several bam files from unknown origin. I'd like to know what software was used to obtain those bam files, e.g. bwa-mem. Is it possible to somehow check it?
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Quantify low complexity regions in DNA sequences

I have fasta files with multiple sequences. They are reads that mapped to the genome outside of probe-targeted regions. From a quick perusal, they appear to be repetitive and have low complexity. Is ...
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How to rename Read Groups

I periodically run into problems with Read Groups. I am in a desperate need for a tool that would allow me to fix them. This time, when I was mapping reads, I formatted all the information in the Read ...
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1answer
403 views

What is "unmapped read segments" in the output of samtools idxstats?

samtools idxstats produces a four column output (see here) ...
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48 views

Reads count in metagenomics

Background: I am developing a pipeline for metagenomic studies of human gut microbiote. In particular, I am mapping the reads data originating from shotgun whole genome sequencing onto a gene ...
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22 views

How to control/normalize for number of reads when calling SNPs using RNA-Seq?

I used the GATK pipeline to call SNPs on males and females using RNA-Seq data. But the males have a higher read count (~43-46M reads) than the females (~40-42M reads). This causes SNP counts to be ...
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115 views

Contamination on genome assembly

I had a question for the community. I have a genome of a new species that has been sequenced via 150pb Illumina paired-end. To verify the quality of the assembly I used the ...
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198 views

Difference between paired-end, mate-pair and long read

I writing here because I have some questions for you. I wondered what the essential differences were between paired-end, ...
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82 views

Question: Bismark methylation for non-uniquely mapping BS-seq reads

I am looking for the DNA methylation, specifically inside small RNAs; piRNA. I am mapping BS-seq reads on the genome with bismark. From the user guide of bismark, it maps reads uniquely on the genome, ...
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34 views

Assemble reads for a specific gene

I have a lot of unassembled sequencing data and I want to build phylogeny for some genes from these data. I can retrieved the sequene by mapping reads to a reference sequence but if the two species ...
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61 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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2answers
733 views

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
203 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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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
181 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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242 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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1answer
50 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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961 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
48 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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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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1answer
202 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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288 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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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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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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844 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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125 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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1answer
119 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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129 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
1k 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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806 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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4answers
281 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 ...
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1answer
206 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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3k 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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33 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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1answer
96 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
160 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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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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138 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
2k 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
3k 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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2k 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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512 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 ...