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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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Where to obtain fastq_illumina_filter

I'm setting up a snakemake pipeline for my lab, and I'd like to install fastq_illumina_filter. All links I can find point to this address for info on it, but the website seems to be down. Is there ...
Whitehot's user avatar
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3 votes
2 answers
51 views

Issues with adapter trimming (Trim Galore)

I am new to coding and especially new to bioinformatics so I am sorry if this is a dumb question. Nevertheless, I am attempting to run Trim Galore! to trim my paired RNAseq data, there are no error ...
tphinney's user avatar
3 votes
1 answer
64 views

RNA-seq QC and alignment error in script

I am analyzing bulk RNA-seq data for Paired-End. I have separate scripts for fastqc, STAR & qualimap but want to run them in a single script which looks like this USAGE: sh rna-qc.sh <path/to/...
S_Malik's user avatar
  • 61
2 votes
1 answer
21 views

GBS- 'Failure' in, Per base sequence content, Sequence Duplication Levels, and Adapter Content

I have a GBS sequence on the Illumina platform that in the FASTQC quality report has “Failure” in : Per tile sequence quality, Per base sequence content, Sequence Duplication Levels and Adapter ...
Gabriela Carvalho da Silva's user avatar
1 vote
0 answers
10 views

How to extract the mutations specific to cancer after variant annotation in NGS analysis

I am working on B cell lymphoma of dogs to identify the specific type of mutations related to this disease. I am performing NGS whole genome sequencing analysis. I have performed the variant ...
Navya Vadlamudi's user avatar
2 votes
1 answer
30 views

How much data to expect from metabarcoding?

I am about to conduct my first metabarcoding study. We will be using the ITS amplicon to look at fungal community composition in soil samples. I need to write a data management plan before I actually ...
Rendzina's user avatar
0 votes
1 answer
17 views

SNP Signatures with Limited WGS Data:

On 80 WGS samples, I'm dissecting SNP signatures linked to milk production in a scarcely studied animal. Post-variant calling and QC association analysis have been tricky. I'm here to tap into our ...
M.Bioinfo's user avatar
  • 376
5 votes
1 answer
75 views

Can index hopping lead to more reads in samples?

We run multiple samples for sequencing on an Illumina NovaSeq machine. After converting the files to fastq format using bcl2fastq, we can see that we have some ...
Assa Yeroslaviz's user avatar
2 votes
0 answers
48 views

I'm trying to run aTRAM tool for assembly, but i'm stuck on this error:

Code: ...
Ro S's user avatar
  • 21
4 votes
1 answer
93 views

split fastq file containing a sequence block at different locations

I have some fastq files (obtained from nanopore sequencing) that contain reads that can be of either of these 5 forms: a known CDS with 3'UTR: ...
jetpacks_reno's user avatar
2 votes
1 answer
34 views

Approaches for filtering bacterial/fungal contaminant sequences from RADseq results

I'm working with RADseq data (288 compressed FASTQ files, *.fq.gz) from 3 plates of plant tissue. I've done demultiplexing and preliminary QC (using the Stacks <...
akoontz11's user avatar
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0 votes
0 answers
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Suggestion for way foward with upregulated genes

I am working with single cell RNA data- control and treated. I analyzed and got a list of upregulated genes. Upon doing pathway analysis all the genes were of apoptotic pathway. But these genes can ...
subhiksha sundaram's user avatar
1 vote
1 answer
73 views

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 ...
subhiksha sundaram's user avatar
0 votes
1 answer
31 views

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....
Shafayet Rahat's user avatar
2 votes
1 answer
89 views

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.
Mudith Ekanayake's user avatar
3 votes
1 answer
254 views

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 ...
Vijith Kumar V's user avatar
3 votes
0 answers
80 views

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 (...
InterestingQuestions44's user avatar
0 votes
1 answer
79 views

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: ...
InterestingQuestions44's user avatar
1 vote
1 answer
76 views

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 ...
Sina Asadi's user avatar
1 vote
1 answer
70 views

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 ...
TheNumber23's user avatar
1 vote
0 answers
22 views

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 ...
S_Malik's user avatar
  • 61
2 votes
0 answers
27 views

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 (...
kane9530's user avatar
  • 181
2 votes
1 answer
314 views

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 ...
Ahsan's user avatar
  • 31
-1 votes
1 answer
53 views

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 ...
Denise Lavezzari's user avatar
0 votes
1 answer
129 views

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 ...
InterestingQuestions61's user avatar
1 vote
1 answer
195 views

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 ...
InterestingQuestions61's user avatar
1 vote
1 answer
701 views

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 ...
InterestingPenguin80's user avatar
1 vote
0 answers
15 views

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 ...
InterestingQuestions61's user avatar
1 vote
1 answer
518 views

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: ...
justinian482's user avatar
2 votes
1 answer
76 views

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 ...
user avatar
1 vote
1 answer
58 views

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 ...
Илья Бетяев's user avatar
1 vote
1 answer
1k views

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 ...
Jackson Xavier's user avatar
2 votes
0 answers
152 views

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 ...
Manu_sab's user avatar
2 votes
1 answer
119 views

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 ...
App.vsh.io's user avatar
2 votes
0 answers
92 views

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: ...
Aranyak Goswami's user avatar
2 votes
0 answers
27 views

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 ...
A.Dumas's user avatar
  • 497
1 vote
1 answer
74 views

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 ...
Burak Muhammed ÖNER's user avatar
1 vote
1 answer
103 views

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,...
David Khutsishvili's user avatar
3 votes
0 answers
32 views

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 ...
Caterina's user avatar
  • 307
1 vote
1 answer
41 views

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 ...
Caterina's user avatar
  • 307
2 votes
2 answers
134 views

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 ...
Caterina's user avatar
  • 307
2 votes
1 answer
164 views

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 ...
justinian482's user avatar
1 vote
0 answers
42 views

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 ...
Shakunthala's user avatar
2 votes
3 answers
97 views

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 ...
Wei Feng's user avatar
2 votes
1 answer
345 views

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 ...
twinkle's user avatar
  • 41
2 votes
1 answer
707 views

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, ...
Oludhe's user avatar
  • 21
2 votes
2 answers
238 views

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 ...
Gustavo de Miranda's user avatar
1 vote
2 answers
98 views

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/...
Adamm's user avatar
  • 206
4 votes
2 answers
140 views

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 ...
Karol Jacek's user avatar
0 votes
1 answer
217 views

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 ...
Moo's user avatar
  • 127

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