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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issue with rna seq analysis
I am working on RNA seq analysis and I would like to know the following things:
Current Methods:
downloaded genome fasta file for non-coding rna from ensembl and got the gtf file for hg38
performed ...
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Generating simulated gold standard NGS dataset/ Any publicly available smal gold standard NGS dataset
I am trying to check my variant generation pipeline for humans. The pipeline is for NGS. I am searching for any dataset(.fastq+.bam+.vcf) for any specific sample. Now, GIAB has a nice dataset for that....
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How to interpret a GenomeScope plot?
I am working on a new sponge genome and I have produced the genomescope plot from the 21-mer kmer frequencies. I have hard time interpreting the plot. Can someone please help me?
Thank you.
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Trimmomatic QC report shows drop in the reads and presence of overrepresented sequences
This question was also asked on Biostars
I am performing a de novo genome assembly using Illumina paired-end short reads, sequenced on a NovaSeq X by our collaborator at UCLA.
At present, I am in the ...
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Issue creating CNV plot from WES data
I received Whole Exome Sequencing data from an NGS company (CARIS, specifically). I received R1 and R2 FASTQ files, a BAM file aligned to hg38, and a VCF file.
I used CNVPytor to create a CNV plot (...
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Invalid tag name: "1KG" and Invalid character '/' in 'SA' FORMAT field at chr1:6197766
I have a VCF file that is of the following format:
...
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What are the output files of RNA-Seq from facility?
This question was also asked on Reddit
I am new in our lab and I am going to do bulk RNA-Seq. What type of files will we get from the company (Genewiz)? Will it be a bunch of Fastq files? or they give ...
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Choosing the best number of species for assay
I am doing amplicon sequencing of a virus across many different regions. Lets say I have 20k unique variants of the same virus that I put into my pcr assay and after sequencing and amplifications I am ...
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What statistical analysis should I perform on DNA variants from unequal number of case (34) and control (11) samples?
I have performed NGS on 34 patient samples and 11 controls. Now I have variants from both groups but there is obvious difference in number of variants.
How can I compare variants of both groups ...
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Off-target % for whole-exome sequencing panel
My samples have been sequenced with the Twist Exome 2.0 sequencing panel, and I want to assess how efficient the sequencing has been. By efficient, I mean examining metrics such as the uniformity (...
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calculate mismatch frequency/rate from a BAM file
I am working with PAR-CLIP data where experimental procedure induces T to C mismatches. The other types of mismatches we can consider coming from artifact such as PCR errors. It could also be an ...
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Variants list to aminoacids
I have the sequencing data of a Monkeypox virus sample. I have the list of variants extracted from Nextclade and I want to find the corresponding aminoacids substitution.
For example I have the ...
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87
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How do I build the MinusB database for Kraken2? (Taxonomy issues)
I am attempting to build my own custom database for Kraken2. I have two questions:
If I have the MCPyV genome in a file called MCPyV.fasta, how do I build a database with just this?
How do I build ...
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Kraken2 Standard Database failing to build (unexpected FTP path)
I am attempting to build just the standard Kraken2 Database by using the following command:
kraken2-build --standard --threads 24 --db $DBNAME
I am returned the ...
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How do I build the "Standard Kraken 2 Database"?
I am at this point in the GitHub tutorial: https://github.com/DerrickWood/kraken2/blob/master/docs/MANUAL.markdown#standard-kraken-2-database
It says to create the standard Kraken database, I use the ...
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DNASTAR viral-host integration assembly keeps failing
I have two NGS files from an NGS company corresponding to the sequencing data from a tumor sample as follows:
TB_7710391_R1.FASTQ.gz
TB_7710391_R2.FASTQ.gz
I have downloaded the genome for MCPyV as ...
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Estimate insert size for single-end data with picard CollectInsertSizeMetrics
I have a BAM file generated from single-end data and I want to estimate the insert size using picard's CollectInsertSizeMetrics as follows:
...
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Sanger sequencing annotation error
I am a student in a Cancer lab. Working with sanger is new to me. While analyzing a report we found an insertion that has not been reported in any databases so far, we were working on checking if the ...
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Problems with BED format for ClinCNV
I have some problems with ClinCNV(https://github.com/imgag/ClinCNV):
I made a BED file from a BAM, then i made a GC count with ngs-bits (link: https://github.com/imgag/ngs-bits )
The main idea of ...
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Need to convert .bed to .vcf. Can the reference build (and necessary .fasta) be determined for a .bed file?
I have some .bed files (and .bim and .fam files) containing data for a number of different samples, and I need to convert them to .vcf.
I've found bed2vcf which is ...
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BAF and LRR calculation and usage
I'm working with a vcf file, in particular I'm trying to identify a gene duplication in a sequenced genome.
I studied bcftools cnv command, but for this tool is needed a vcf file containing a BAF and ...
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Is it common to get different number of SNVs+Indels across samples from vcf files generated using GATK and DRAGEN (counts are higher for GATK)?
We have 2 vcf (Whole Exome Sequencing (WES) data; germline samples) files (e.g., vcf_1 and vcf_2). vcf_1 was generated (Ref. genome: hg38) using the GATK pipeline for 250 children and their parents ...
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Adapter trimming
I am trying to do adapter trimming, alignment and sorting for a range of large scale paired end fastq files. The code I am using is given below:
...
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What data set was used Gene Expression Data of Postmortem Tissues?
I want to run the experiments "Sample Application to Genotype-Tissue Expression (GTEx) Project Gene Expression Data of Postmortem Tissues" mentioned in "Enter the Matrix: Factorization ...
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Expression analysis of miRNAs with normal RNA-seq data without small RNA-seq data
I am looking to perform expression analysis of miRNAs with normal RNA-seq data lacking small RNA-seq data?
Which path should I choose for known miRNAs and unknown new miRNAs?
Data set: rna-seq data ...
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Lower case vs. upper case nucleotids in sequence vs. dots at the end
What is the difference between lower case and upper case nucleotides in a sequence? My other question is what are the dots at the end of the sequence? Some examples are shown below:
GGgG,GGgG,GGgG,...
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Can multiplexing in Sequel II SMRTcells reduce the coverage?
I would like to have high depth of coverage of the transcriptome, but I need to analyze several tissues. I was suggested to pool all 5 tissue samples in same SMRT 8M cell. Will this result in a ...
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How can Iso-Seq reverse transcriptase artifacts be avoided?
My end goal is annotate a de-novo assembled genome. When trying to select the best method for transcriptome assembly I read Iso-seq was the preferred method. However other people suggested Nanopore as ...
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What is the most accurate approach for de Novo sequencing?
I'm trying to decide between PacBio HiFi or Illumina sequencing platforms for sequencing the genome of aChrysina scarab. We want to identify the color pattern loci and also perform a phylogenetic ...
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Count reads at specific gene features
I have BAM files from an RNA-Seq experiment and for all genes (or a subset) I want to get the number of reads in regions around the TSS (e.g. 2kbp) and the TES (e.g. 2 kbp) and calculate the ratio ...
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System specifications for NGS data analysis
I have a 3.7 GB whole genome data of a eukaryote, for which genome assembly, gene prediction, and annotation steps have to be performed. In some time I would also need to analyze the transcriptome ...
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Can we do NGS library preparation using UMI with large amount of DNA input?
We know that in next-generation sequencing (NGS), the unique molecular identifier (UMI) can reduce or eliminate sequencing or PCR errors and result in very high accurate data. Therefore, UMI is widely ...
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How can I generate ASV file from nanopore sequencing data?
I am converting the fast5 file to fastq by using guppy basecaller after that by using kraken2 classified the sequence.
Now I am trying to generate ASV file.
Is this possible to generate ASV file from ...
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Extracting base sequences from ABI/AB1 sanger sequencing chromatogram
I am trying to understand the sanger sequencing ABI/AB1 file format better, and extract base calls from given signal intensities over time.
As I understand, reading in a raw AB1/ABI file into python, ...
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BWA-mem and sambamba read group line error
this question has been asked [and answered] on Stack Overflow
This is a two-part question:
help interpreting an error;
help with coding.
I'm trying to run bwa-mem ...
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How can I get latest .vcf files with annotation data?
I'd like to perform the annotation (1000genomes, COSMIC etc.) of my variants using SnpSift and SnpEff, however so far all I get are vcf files for separate chromosomes:
http://ftp.1000genomes.ebi.ac.uk/...
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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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Why must a maximal non-branching path be a contig?
The following is from Bioinformatics Algorithms:
Fortunately, we can derive contigs from the de Bruijn graph. A path in a graph is called non-branching if in(v) = out(v) = 1 for each intermediate ...
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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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Locate the saved template of minknow
I am using minknow on ubuntu. After saving the template (settings), where is that file locally stored in the system?
Also what is its format/extension?
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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 ...
M__♦
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What are Dummy fastqs?
I had to do a file transfer for in our system to run from fastqs and I came across the term "dummy fastq", not sure what exactly it means and what is the purpose of it in the workflow. Can ...
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How to retrieve fasta sequence after local blast?
I have created a Blast database using a reference genome. Then, I have performed a local blast search in command line using a gene of interest. I have obtained some hits with the usual Blasting ...
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Drug-DNA complex simulation using AMBER
I want to carry out the simulation of drug-dna complex by placing the drug molecule at particular site in my DNA sequence (intercalating site). I have PDB files of both drug and DNA sequence. I will ...
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When using cutadapt, can I specify an R2 adapter as optional when I have a required R1 adapter?
I'm using cutadapt 3.5 to trim adapters and perform some filtering on paired-end data.
Both R1 and R2 sequences have 3' adapters that might be found depending on the sequence length, but R1 also has ...
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Shotgun analysis: Where and how to start?
I am a complete rookie in shotgun analysis, having only performed marker gene analysis before. I have a pipeline that I want to try but I can't wrap my head around how it all works.
Please correct me ...
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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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