Questions tagged [ngs]

use for general applications of high-throughput nucleotide sequencing methods that use short-read technology (e.g. Illumina, IonTorrent). For long-read sequencing, use 'long-read', or a more specific tag if applicable (e.g. nanopore, pacbio).

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Does the number of RNA reads per cell obtained from the 10X scRNA experiment depend on amount of mRNA in given cell?

As we know, the amount of RNA reads per cell obtained from 10X scRNA experiment vary between cells. I wonder if this is effect of technical issues or does the number of RNA reads per cell obtained ...
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Reference variant detected as altered one in bam file

I received (from manufacturer) several .bam files and I used four callers (samtools, freebayes, haplotypecaller, deepvariant) to find some sequence variants. In obtained .vcf files, I took a closer ...
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Cross-checking metagenomics analysis via reference genomes

I'm looking at verifying metagenomic pipeline output for small genomes (viruses) on short read data. Background Most of the work in small-genome metagenomics is performed using bacteria and I'm ...
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A question on Homer normalization

When using annotatePeaks.pl script from Homer software to create histograms, the output is normalized per bp per peak (on top of normalization to 10 million tags). What does it mean to normalize per ...
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Metagenomics pipeline recommendations for short-read data

de novo metagenomics on viral NGS data is a hot-topic. On this site alone at least 4 specific algorithms have been used to identify multi-strain/multi-species for a given data set, however these do ...
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How to customise options.json and config files to run WDL workflow in a remote server (not cloud)?

I am running wdl workflow (ExomeGermlineSingleSample) with Cromwell locally. Based on here (https://cromwell.readthedocs.io/en/stable/RuntimeAttributes/), I have set "backend": "Local&...
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Dictionary and index of vcf for base recalibration step

I need suggestions in creating index and dictionary with vcf files. For the base recalibration step, I downloaded Homo_sapiens_assembly38.known_indels.vcf.gz from the given link: https://console.cloud....
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Why is bcl2fastq2 taking so long to calculate stats?

Our lab has been using bcl2fastq v2.20.0.422 to demultiplex RNA-seq data sequenced on an Illumina Novaseq machine on a beefy EC2 instance and we've run into the strange problem: namely that while ...
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BWA-MEM2 alignment-Snakemake

I have started using snakemake 6.5.2 to align fastq files with reference file. I have pasted the error below in this question. How to allocate memory in the snakefile and read the header from samfile, ...
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ClinSV v1.0 has issue with hg38

I followed all the steps to run the software on the server but I get that the lumpy-sv does not work with clinsv v1.0 ...
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Output of allelic association doesn't write the rsID

I'm using plink (1.9b5) to do allelic association. My problem is that my output does not write the rsID (SNP), it just writes a dot. Output: ...
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Illumina vs PacBio vs Nanopore price

Can someone help me with comparing the cost per run of Illumina, PacBio and Nanopore sequencing technologies?
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RNA_Seq data aligned used uniquely or multi mapped reads impact on result interpretation

I have some transcriptomic (Whole) sequencing data that I should analyse. I would like to do raw data alignment to a reference genome taking into account the multi mapped reads and uniquely mapped ...
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fastq file format unknown

I have paired-end fastq files some of which seem to be in a weird format (from a collaborator, not a public database). When I cat the file I get what seem to me to ...
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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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Supplementary aligments in VAF

This question has also been asked on Biostars I have a doubt, are supplementary alignment usually considered when the variant allele frequency is calculated? Thanks a lot. In my case I have some ...
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1 answer
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How to generate a consensus sequence from a multi-reference BAM file?

I am trying to generate a consensus sequence from a BAM file that was generated by mapping reads to a reference FASTA containing multiple sequences. Usually, I generate consensus sequences from BAM ...
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Calling variants with DeepVariant on targeted NGS sequencing (custom library)

I am seeking advice regarding DeepVariant analysis. To avoid false positives I'm using several variant callers and then the resulting common set will be considered as TP variants. One of the callers ...
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Genomic relationship matrix explanation

I am seeking the definition of the genetic distance matrices used in genomic prediction. What is the difference between a Rodger distance and kinship matrix? what people mean when they talk about ...
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Template files for bioattributes and meta-data [closed]

I am in process of submiting my FASTQ files to the SRA database. I am a little confused about how to fill the biosample attributes and SRA metadata. I would really appreciate if you anyone could share ...
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1 answer
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Trimmomatic trims most of the reverse strand

I have a paired-end data. It is from small part of human genome not a WGS or WES. I use BWA for alignment and do variant calling. Since I had some false positives I wanted to do trimming. I used ...
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Inheritance annotation for nsSNPs

I am relatively new to SNP analysis. Is there a database to find annotation about the Mendelian inheritance of SNPs? I have a small list of nsSNPs (non-synonymous SNPs) and I need to find how they are ...
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Need a alternative or more complex version of venn diagram in python for matching dna sequences

I am in google colab and I have combined and set up a data frame list of sequences that goes like this Location ID Sequence 1.1 ........ A ........ AAGAGATA 1.2 ........ A........... ...
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2 answers
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Error after trimming illumina adapters

I am removing illumina adapters of the NGS data with a loop. My NGS data is storage in /data/HTS_seq/. I used this function: ...
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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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snakemake - using output file not explicitly declared as input for subsequent rule

I am writing a snakemake that, up to now should use raw sequencing reads to produce a draft assembly using canu use minimap2 to align the raw reads against the draft assembly, generated by canu. ...
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How can I extract all known mutations of my BAM (or SNP/INDL files)?

I am using a Genome Explorer tool to see all the mutations on my own DNA. My particular interest is on listing the variants and get their names/ids. Here are a few screenshots: You can see that ...
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1 answer
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Blast results filters

I performed a blastn search of NGS data against ssRNA database download from Internet, with a expected value 10-4. The size of NGS data reads is of 125 bp. I have analyzed the blast results of the ...
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2 votes
2 answers
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How to remove sequence reads contains more than 2 X from multifasta file?

I have 5000 protein sequences in one multifasta file. I found more reads have gaps as X in their reads. So, want to eliminate those reads completely (Whole protein seq) from the file. I am keeping ...
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1 answer
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No MQ tags in VCF files

To call minority variants in my Mtb sequences I'm using a pipeline of ...
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1 answer
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How can I find the position of every mutation where the Allele Frequency is greater than X, in regards to a reference such as Hg19?

I have a Human genome sequenced in a BAM file (along with other files with the indels, snps, cnvs). I want to find every mutation with regards to the reference Human genome. However the majority of "...
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Generate VCF from different .bam files with different chromosome names

I have two resources of .bam files. One is generated by our lab (1 sample = 1 bam). One is downloaded online (again 1 sample = 1 bam). For the downloaded samples the chromosomes are labelled: chr1, ...
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1 answer
315 views

How to get the intersection of peaks after peak calling using MACS2?

I have following 5 narrow peak files after peak calling. K14_peaks.narrowPeak K15_peaks.narrowPeak K16_peaks.narrowPeak K3_peaks.narrowPeak K8_peaks.narrowPeak I ...
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Downloading dataset from SRA (SOLiD Platform)

Trying to download colorspace data from SRA, but getting an error abi-dump -A SRR1657115.sra abi-dump.2.9.6 err: item not found while constructing within ...
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2 votes
1 answer
130 views

Any user friendly way to find rare mutations in whole genome raw?

Is there any user friendly way to find rare mutations in the individual human whole genome sequencing raw data? (from Dante, 30x coverage). To be more specific, I want to find mutations from this ...
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1 answer
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Add new tool to galaxy

I am trying to include a new tool to the galaxy main menu, following these instructions. tool_conf.xml doesn't exists only ...
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3 votes
1 answer
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How can I export a full alignment from IGV as an image?

The IGV browser lets you export an alignment as an image (File => Save Image). However, this image only contains those reads that fit in the viewing window: As you can see in the image above by ...
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1 vote
2 answers
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Extract reads from bam files by their @RG

How could I extract reads from bam files by their read groups @RG ?, I've got one file containing all reads for 5 samples, the SM is appears NONE, So, I want to extract each sample reads by the @RG ...
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5 votes
1 answer
851 views

PCA vs tSNE in single cell RNA-seq

What makes tSNE being the preferred dimensional reduction for visualization in single cell RNA-seq over PCA? I am aware that tSNE works better at showing local structures and fails to capture global ...
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2 answers
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Can we use GTEx data as control data for TCGA data?

I am using Recount2 TCGA data and was wondering is it right to use GTEx data as control data for this. I would really appreciate your views on this?
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2 votes
5 answers
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Testing for viral/bacterial infection FASTQ files

What's the best way to test if a pair of Illumina FASTQ files from blood DNA extraction contain any reads are from viral or bacterial infection from human samples? Thanks in advance. EDIT: Looking at ...
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1 vote
2 answers
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VEP output SIFT_score unclear

We have been experimenting with VEP (Variant Effect Predictor). One of the meta data attributes that we are interested in is the SIFT score, indeed when we apply the dbNSFP plug we get a column ...
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2 votes
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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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3 votes
3 answers
265 views

Tools for quality trimming at 5 prime?

I'm looking for a tool that is capable to do quality trimming at 5' end and it is configurable. For example, I can choose the quality trheshold, the read length, etc. Any recommendations? I'm ...
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amplicon sequencing set up

I have .fastq files from a targeted amplicon sequencing by Illumina MiSeq run. I want to get the target sequences to perform Blast search on them. What is the pipeline to use for processing amplicon ...
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2 votes
2 answers
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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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4 votes
1 answer
99 views

Simulate and test CNV workflow?

I'd like to evaluate a CNV project. My aim is to evaluate if the scripts are sufficient for calling reasonable CNVs. I know they have a paper, but their scripts may be buggy... and all papers are ...
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3 votes
1 answer
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Why is the total read number still more than the paired in sequencing after removing the duplicate in samtools flagstat output?

After alignment using BWA, I have removed the dupliment using the samtools(Version: 1.9). My procedure is as follows: ...
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2 votes
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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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