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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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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1answer
42 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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1answer
17 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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37 views
bcftools mpileup double counting for overlapping reads
I have a dataset of paired-end reads, which can overlap. So when i run bcftools mpileup this overlap gives a double count in the depths so we see DP = 2 even though it should be DP = 1.
The question ...
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1answer
63 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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3answers
48 views
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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26 views
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
194 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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1answer
28 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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20 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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2answers
107 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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1answer
126 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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1answer
64 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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1answer
33 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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1answer
57 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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20 views
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
534 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
151 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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44 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
143 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
202 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
41 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
824 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 ...
2
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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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125 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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1answer
178 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
248 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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91 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
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?
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1answer
709 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
106 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
105 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
123 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 ...
2
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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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2answers
692 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
41 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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4answers
255 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
193 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
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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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1answer
2k 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
32 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
95 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
146 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
992 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. ...
2
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2answers
106 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
1k 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 ...
3
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1answer
1k 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
44 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
494 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 ...
