Tag: MAPQ

Accepted bedtools 2.31.1+dfsg-2 (source) into unstable

—–BEGIN PGP SIGNED MESSAGE—– Hash: SHA512 Format: 1.8 Date: Mon, 04 Dec 2023 20:13:11 +0100 Source: bedtools Architecture: source Version: 2.31.1+dfsg-2 Distribution: unstable Urgency: medium Maintainer: Debian Med Packaging Team <debian-med-packag…@lists.alioth.debian.org> Changed-By: Étienne Mollier <emoll…@debian.org> Closes: 1043957 Changes: bedtools (2.31.1+dfsg-2) unstable; urgency=medium . * d/clean: new: remove test/getfasta/t.fa.gz.gzi. (Closes: #1043957)…

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Mapping quality in `vg giraffe`

Mapping quality in `vg giraffe` 0 I ran vg giraffe to get the GAF file. I have some questions about the 12th column, that is mapping quality(MAPQ). The difference of MAPQ standard between aligners makes me confused. I found MAPQ is in the range[0, 60] which similar with bwa-mem, but…

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Chromatin priming elements direct tissue-specific gene activity before hematopoietic specification

Introduction The development of multicellular organisms requires the activation of different gene batteries which specify the identity of each individual cell type. Such shifts in cellular identity are driven by shifts in the gene regulatory network (GRN) consisting of transcription factors (TFs) binding to the enhancers and promoters of their…

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Find all locations of aligned reads with MQ (Mapping Quality) = 0.

Find all locations of aligned reads with MQ (Mapping Quality) = 0. 1 I have white reads in BAM file with MQ = 0. I know that it means that reads align to multiple locations. I know location of one align, but can i find another location/locations in IGV? If…

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Problem aligning target capture sequencing of a few hundred regions to the human reference genome

Hello, We have been trying to develop a target capture sequencing of around one hundred regions in the human genome. The probes capture a region of approximately 500bp around some genetic variants of interest. Our bioinformatics pipeline uses bwa-mem for aligning the captured reads to a custom genome reference. These…

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Filtering for primary and secondary reads using sam flags (0 properly paired reads in alignment step)

Hey everybody, I have just performed the alignment of paired-end reads to a reference using bwa mem with the -M flag, ran samtools markdup and flagstat. Flagstat produced the following output: 4985084 + 0 in total (QC-passed reads + QC-failed reads) 1806492 + 0 primary 3178592 + 0 secondary 0…

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fragments file generation via Sinto from CellRanger output

fragments file generation via Sinto from CellRanger output 0 Hi, I am following the instructions for the PASTA package (satijalab.org/seurat/articles/pasta_vignette.html). This package uses scRNA-seq data to infer alternative polyadenylation usage from scRNAseq data. It requires among many input files also a fragment file. The authors state the following must be…

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Pysam pileup and Rsamtools pileup output discrepancy

I have mRNA sequencing data that I’ve aligned to a genome. Specifically, I am interested in determining the total number of reads at each base and the percentage of occurrences of A, T, G, C, deletions, and insertions at these bases. I have utilized both pysam pileup and Rsamtools pileup…

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ERROR::INVALID_ALIGNMENT_START Alignment start should be 0 because reference name = *.

The following code I have developed with PYSAM looks for reads completely soft clipped import pysam import re import sys import os if len(sys.argv) != 2: print(“Usage: python improve_bam.py input_bam_path”) sys.exit(1) # Input BAM file path provided as a command-line argument input_bam_path = sys.argv[1] # Generate the output BAM file…

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Mapping of paired-end ddRADseq results in 0.00% of reads pairing

Hey all, I’m trying to map my RADseq to a reference genome, and none of my paired-end reads are being paired. Forward and reverse reads are both mapping separately, but not pairing. This problem is consistent for all of my samples. Any help would be much appreciated!! I also viewed…

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Cost-effective DNA methylation profiling by FML-seq

The FML-seq method The protocol for FML-seq comprises only three steps (Fig 1A). First, genomic DNA (gDNA) is digested by a methylation-dependent restriction endonuclease that cuts at a certain distance from the 5-methylcytosine or 5-hydroxymethylcytosine in its motif and leaves a short overhang (10). Second, a master mix is added…

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Scaling logical density of DNA storage with enzymatically-ligated composite motifs

Composite motifs as building blocks for DNA storage A composite motif is a representation of a position in an oligo sequence that uses a combination of motifs drawn from a fixed motif library to encode data. For example, assuming a library of 32 motifs, and a combination factor of four,…

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Cell-free chromatin immunoprecipitation to detect molecular pathways in heart transplantation

Abstract Existing monitoring approaches in heart transplantation lack the sensitivity to provide deep molecular assessments to guide management, or require endomyocardial biopsy, an invasive and blind procedure that lacks the precision to reliably obtain biopsy samples from diseased sites. This study examined plasma cell-free DNA chromatin immunoprecipitation sequencing (cfChIP-seq) as…

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BiocMAP: a Bioconductor-friendly, GPU-accelerated pipeline for bisulfite-sequencing data | BMC Bioinformatics

Overview In the first alignment module, FASTQ files are checked with FastQC 0.11.8 [15], and by default samples that fail the “Adapter Content” metric are trimmed using Trim Galore! 0.6.6 [16] (see Methods: Configuration for alternative trimming options). The resulting FASTQ files are aligned to a reference genome using the…

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Single-cell massively-parallel multiplexed microbial sequencing (M3-seq) identifies rare bacterial populations and profiles phage infection

Bacterial strains and growth conditions for eBW1 B. subtilis 168 and E. coli (MG1655) were streaked out from a frozen glycerol stock onto an LB plate and grown overnight at 37 °C. Following a night of growth, a single colony was picked and inoculated into 5 ml of LB broth and grown…

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Why extracting reads from BAM to fastq produces small amount of reads?

Hi, I got a peculiar issue with one of my datasets – when I try to extract reads from a BAM file (which has 20 mil reads) to a fastq – I get only ~400k reads. Here is what I do: I first sort the bam by name: samtools sort…

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Plotting Genomic Overlaps with GRanges

I have come up with two approaches using two different findOverlaps methods (Genomic Alignments R package) for which I have opted to use subsetbyOverlaps as this suits my needs better. I’m trying to plot the genomic coordinates that overlap, was trying with the GViz package but not sure if this…

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Filtering unmapped reads from BAM files

Filtering unmapped reads from BAM files 0 I am applying the GATK best practices for variant calling pipeline. By applying the picard addOrReplaceReadGroups I got the error: “invalid_mapping_quality”, I read the post Final Solution For “Mapq Should Be 0 For Unmapped Read.”. So decided to remove unmapped reads with samtools…

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.bed files from sequencing platform not containing intervals of “alt”, “random” haplotypes. How do I perform coverage and haplotype caller?

.bed files from sequencing platform not containing intervals of “alt”, “random” haplotypes. How do I perform coverage and haplotype caller? 0 Hello. I’m building my first human exome variant call pipeline, and I’m learning the basics. I encountered this issue for the first time when trying to obtain a per-base…

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VG Giraffe multi-mapped reads and definition for MAPQ score

VG Giraffe multi-mapped reads and definition for MAPQ score 0 Hi, I am recently playing with VG giraffe. The tool is amazing. I want to say thank you for the developers of VG team. I do have 2 little questions regarding the alignment. What is the definition of MAPQ (mapping…

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BAM file contains much more reads than expected

Hi, I have a question regarding mapping qualities and multimapping reads. I’m working with the Allen Brain Atlas TBI data, which can be found at aging.brain-map.org/rnaseq/search. I’m confused about the different numbers provided. Taking the first sample as example. According to the provided data: rnaseq_total_reads: 32,275,545 rnaseq_percent_reads_aligned_to_mrna: 31.7% rnaseq_percent_reads_aligned_to_ncrna: 7.11%…

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Filtering mitochondrial reads from ATAC-Seq aligned reads- what to do with reads that have MT in RNEXT field

Hi all, I am trying to filter mitochondrial reads from my ATAC-seq data after trimming with Trimmomatic and then aligning with Bowtie2. After searching through many pipelines, I have found 2 ways that people often do this (both using inputs that are sorted and indexed BAM files): 1) with samtools…

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Reads with highest MAPQ values from SAM files are showing mismatches to reference sequence and IGV classified them as supplementary reads

Hi all, I am expressing a GFP synonymous variant library in human cells and sequencing its RNA on the nanopore and I am having some trouble analysing the data. Initially, I basecalled all the fast5 files using the super accuracy model in the guppy basecaller, then I discarded the reads…

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minimap’s SAM file MAPQ value for the unique alignments

minimap’s SAM file MAPQ value for the unique alignments 0 Hi, I have mapped my reads from nanopore RNAseq to the reference database using the minimap. Minimap SAM files have MAPQ values from 0 to 60 in the fifth column. While 0 indicates the reads that can possibly map to…

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Upstream pseudogene causing MAPQ 0 and exclusion during variant calling

Upstream pseudogene causing MAPQ 0 and exclusion during variant calling 0 Hello, just upstream of GBA1 is a pseudogene that is quite similar to GBA1, this makes most of my mapped reads have MAPQ 0 across this region (I think? I use bwa-mem2 with default settings), and the variants are…

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MAPQ filtering for clinical applications

A discussion recently arose about how one ought to filter MAPQ in a clinical setting, i.e., where a NGS sample is being processed in order to produce a result for a patient who has an unknown or hypothesised diagnosis. The result could obviously be key. It was suggested by a…

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High number of duplicates and low percentage properly paired

High number of duplicates and low percentage properly paired 0 I have some paired end sequencing data that I have trimmed using cutadapt. It was sequenced on an illumina novaseq 6000 and is low coverage RADseq data (2-3x). My cutadapt script used forward and reverse adapters from illumina : cutadapt…

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Genecount-difference between HT-seq count, RSEM, and Kallisto

Genecount-difference between HT-seq count, RSEM, and Kallisto 0 Hi I ran three genecount software tools (ht-seq, RSEM, Kallisto) to calculate genecount of RNA-seq data. For Ht-seq, i used STAR aligned Transcriptomesortedcordinate.bam file and defautl MAPQ score with intersection_nonempty mode. For RSEM, i used STAR aligner (used .gtf for building reference)…

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10x Cellranger SN RNA-seq BAM file output looks off

I’m working with SN RNA-seq data that I processed using Cellranger v. 7.1.0 (which uses STAR for alignment with default settings). Using the samtools flagstat command to look at a BAM file output: 284890368 + 0 in total (QC-passed reads + QC-failed reads) 0 + 0 secondary 0 + 0…

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Calling zero mapping quality variant

Calling zero mapping quality variant 0 Hello Is it possible to call variant with read that has zero mapping quality at the region? I found that there is INDEL in my BAM file when I visualize in IGV but the variant is not in gVCF, I have checked the average…

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Can’t convert paired end BAM to bed using bedtools

Can’t convert paired end BAM to bed using bedtools 0 Hello, I got .bam files from my pipeline and i merged them with samtools merge ALL.bam *.bam and I got this % of mapping 3825773 + 0 in total (QC-passed reads + QC-failed reads) 3825773 + 0 primary 0 +…

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fail to open index BAM file”

Dear all, I applied CNVkit to call copy number variations in a case-control study, with vascular malformation samples as case and healthy individuals as control using the following command. /ifs9/B2C_RD_P5/lijia3/CONDA/bin/python /ifs9/B2C_RD_P5/lijia3/CNVkit/cnvkit/cnvkit.py batch /zfsyt1/B2C_RD_P5/lijia3/VM/*.bam –normal /zfsyt1/B2C_RD_P5/lijia3/VM_controls/Control/Control-Kid/*.bam –targets BGI_Exome_V4_kit_200bp.bed –antitargets my_antitargets.bed –annotate refFlat.txt –fasta /ifs7/B2C_SGD/PROJECT/PP12_Project/analysis_pipeline/HPC_chip/db/aln_db/hg19/hg19_chM_male_mask.fa –access access.ucsc.hg19.bed –output-reference VM_normal_adult_reference.cnn –output-dir results_Kid/ –diagram…

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PHG Load haplotype and create consensus

Here, presented my PHG scripts, config, wgs_keyfile. 1. Create valid intervals docker run –name test_assemblies –rm -v /DATA/jysong/PHG/ver1.0_phg/:/phg/ -t maizegenetics/phg:1.0 /tassel-5-standalone/run_pipeline.pl -Xmx100G -debug -configParameters /phg/Masterconfig.txt -CreateValidIntervalsFilePlugin -intervalsFile /phg/inputDir/reference/glyma.Wm82.gnm4.ann1.T8TQ.gene_models_main.bed -referenceFasta /phg/inputDir/reference/glyma.Wm82.gnm4.4PTR.genome_main.fixed.fna.gz -mergeOverlaps true -generatedFile /phg/validBedFile.bed -endPlugin &> Log/1.Create_validinterval.txt & 2. Create initial DB docker run –name create_initial_db –rm -v /DATA/jysong/PHG/ver1.0_phg/:/phg/ -t…

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Interpreting the meaning of outward facing reads

Interpreting the meaning of outward facing reads 0 Sorry i have lots of data and im not sure how to interpret. I took mRNA created by IVT from our plasmid and performed paired end Illumina sequencing.I used samtools stats to get info about the various samples: read length ~150, insert…

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Extracting list of target genes from ChIP-seq bigwig file

Extracting list of target genes from ChIP-seq bigwig file 1 I am reading a publication where the authors performed ChIP-seq on a specific TF. Their data was deposited on NCBI in bigwig format. I would like to extract the final list of target genes. Is this possible using the bigwig…

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Compressing BAM, SAM, CRAM | Genozip

How good is Genozip at compressing BAM files? ​ See Benchmarks. ​ Compressing a BAM, SAM or CRAM file  ​ In the rest of this page we will give examples of BAM files. Genozip is also capable of compressing SAM files, and with some limitations, CRAM files as well. ​…

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featureCounts paired end reads wrongly assigned on the single end mode

featureCounts paired end reads wrongly assigned on the single end mode 0 Hello! I have RNAseq data from samples containing 2 to 3 bacterial strains each. For the analysis I performed FastQC, trimmomatic to remove adapters and then Bowtie2 to align the reads to the reference genomes. I have one…

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Samtools Convert Sam To Bam With Code Examples

Samtools Convert Sam To Bam With Code Examples In this session, we’ll try our hand at solving the Samtools Convert Sam To Bam puzzle by using the computer language. The code that follows serves to illustrate this point. # Basic syntax: samtools view -S -b sam_file.sam > bam_file.bam # Where:…

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different result using minimap2 and pbmm2

Hi all! I am analysing CSS Pacbio data and each sample came from different run, in particular I have three files for each sample. I tested both pbmm2 and minimap2 to align my long reads, after getting the consensus sequences. This is the command I used to run mnimap2: minimap2…

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pjotrp/sambamba – sambamba – Genenetwork

10 years ago ​ 10 years ago ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ 10 years ago ​ ​ ​ ​ ​ ​ 10 years ago 10 years ago 10 years ago ​ ​ 10 years ago ​ 10 years…

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I wonder if someone please explain what secondary, supplementary, duplicates and paired in sequencing mean in samtools flagstat

I wonder if someone please explain what secondary, supplementary, duplicates and paired in sequencing mean in samtools flagstat 0 ”194492 + 0 in total (QC-passed reads + QC-failed reads) 80 + 0 secondary 0 + 0 supplementary 0 + 0 duplicates 193804 + 0 mapped (99.65% : N/A) 194412 +…

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Ubuntu Manpage: sambamba-view – tool for extracting information from SAM/BAM files

Provided by: sambamba_0.8.2+dfsg-2_amd64 NAME sambamba-view – tool for extracting information from SAM/BAM files SYNOPSIS sambamba view OPTIONS <input.bam | input.sam> [region1 […]] DESCRIPTION sambamba view allows to efficiently filter SAM/BAM files for alignments satisfying various conditions, as well as access its SAM header and information about reference sequences. In order…

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sam – Use Htslib to create auxilary tags in bam file C++

I am creating a threaded c++ file where i generate in silico bam files, using header, DNA sequence and read information. First i use bam_init1() to create the bam1_t structure just named “b”. Then i use bam_set1 to create the actual sequence entry in the bam file bam_set1(b,read_id_length,READ_ID,flag,chr_idx,min_beg,mapq,n_cigar,cigar,-1,-1,0,strlen(DNAsequence),DNAsequence,quality_string,l_aux) And finally…

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Samtools flagstat confusing result of a merged bam file

Hi, I am a bioinformatics student and I am struggling with an issue, I had paired-end fastq files for one sample with some low-quality bases at the end and adapter contamination, so I went and I trimmed my reads with trimmomatic, it gave me 4 files that I used for…

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[SOLVED] changing the order of input changes samtools merge ouput

I realized that this is a stupid mistake I have made. Since samtools do not overwrite the files by default, the output that I get from samtools merge output.bam f2.bam f1.bam wan’t what I thought it was below is my original post ++++++++++++++++++++++++++ I’m using samtool/1.9.0 and I’m trying to…

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Overestimation of number of reads from nanopore data (flagstat)

Same issue as mentioned on the minimap2 tool: github.com/lh3/minimap2/issues/236#issue-361097444 For example nanopore reads aligned to the host transcriptome the flagstat output is: 5953480 + 0 in total (QC-passed reads + QC-failed reads) 2961480 + 0 secondary 22696 + 0 supplementary 0 + 0 duplicates 4195469 + 0 mapped (70.47% :…

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Samtools flagstat

Samtools flagstat 1 I aligned my ONT sequencing run with minimap2, subsequently I filtered the file using samtools view -b -F 256 aln_transcriptome_sorted_6.bam -o filtered_aln_transcriptome_6.bam to end up with primary alignments only. When I run samtools flagstat on the filtered file I get the following output: 3502608 + 0 in…

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Primer design for variants in duplicated genes?

The issues you face relate to the fact that the majority of the genome exhibits sequence similarity, i.e., similarity with other regions in the genome. Much of this is indeed related to gene duplication events, with the duplicated genes acquiring new functionality over time due to mutations. As a rough…

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Single-cell DNA and RNA sequencing reveals the dynamics of intra-tumor heterogeneity in a colorectal cancer model | BMC Biology

Organoid culture of small intestinal cells and lentiviral transduction C57BL/6J mice and BALB/cAnu/nu immune-deficient nude mice were purchased from CLEA Japan (Tokyo, Japan). The small intestine was harvested from wild-type male C57BL/6J mice at 3–5 weeks of age (Additional file 1: Figure S9A). Crypts were purified and dissociated into single cells,…

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MAPQ (Mapping quality) of 0 for most reads from BWA-MEM2 (with no secondary alignment or other apparent reason)

Hello, I got a very weird output from BWA-mem2 – most of the reads have mapping quality of 0, even though there is no secondary alignment or anything else suspicious. I got sequencing data that was aligned with Novoalign to hg18, the data was bam files. I needed to realign…

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Output of samtools view, what does the third column actually represent?

The samtools view outputs information from SAM and BAM files in SAM format. You can find a description of the SAM format here: samtools.github.io/hts-specs/SAMv1.pdf Section 1.4 deals with the meaning of each of the manditory coloumns. It includes the following table: Col Field Type Regexp/Range Brief description |—|——|——-|—————————-|—————————————-| 1 QNAME…

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qualimap2 mean mapping quality

qualimap2 mean mapping quality 0 I’ve done a contrast experiment to see the difference between the bam with BQSR and the bam without BQSR. I use qualimap to evaluate both bams. This is the confusing part. Using hap.py and the giab na12878 truth vcf, shows the bam with BQSR is…

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How To Filter Mapped Reads With Samtools

Hi, You get a bam (machine readable sam) file after mapping, and it contains information about mapped and unmapped reads. To get the unmapped reads from a bam file use: samtools view -f 4 file.bam > unmapped.sam the output will be in sam to get the output in bam, use:…

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Calling variants on reads with MAPQ=0 on HaplotypeCaller or bcftools mpileup

Calling variants on reads with MAPQ=0 on HaplotypeCaller or bcftools mpileup 2 I am working with about 500 samples of human exome data. used hg19 to align my reads and ran a standard best-practices GATK workflow. Later only to realise that a small 1Mb loci has not mapped properly due…

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Mapping quality and XS score

Mapping quality and XS score 0 Hi, I am currently looking through bam files using igv to manually check if the mutations not called by Mutect2 are really an error or not. Until now, to filter reads with low quality, I have used only MAPQ > 30 and didn’t consider…

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How to properly combine two bam files of a paired-end data

How to properly combine two bam files of a paired-end data 3 Hi all! I am mapping a paired-end read separately using bowtie2. After that, I want to combine the two bam file into one for downstream analysis. How to properly do this combination? I tried: samtools sort -n R1.bam…

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