Questions tagged [quality-control]

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Is this a complete bacterial genome? (CheckM)

I've been working with a bacterial genome assembly. The initial stats look pretty good (3 main pieces, depth ~50x [20x - 100x] if I calculated it right). I'd expect this to be a virtually complete ...
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  • 595
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2 answers
44 views

What are some ways to check metagenomic bin quality?

I am new to metagenomic binning. I've used CheckM in order to estimate completeness and contamination values, and most of my bins of interest appear to have good values. My workflow was pretty ...
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1 vote
2 answers
174 views

SyntaxError after end of Snakefile code?

I am trying to run Fastqc on multiple files using Snakemake but am receiving an ambiguous SyntaxError past the last line of code in the Snakefile. Snakefile: ...
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0 votes
1 answer
160 views

Snakemake Fastqc: "Multiple run or shell keywords in rule run_fastqc."

I am trying to check the quality of RNA-Seq data from Illumina using fastqc in snakemake in a conda environment. I get the error "Multiple run or shell keywords in rule run_fastqc". ...
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2 votes
1 answer
224 views

What is the right way of calculating a Phred score by hand?

i am trying to calculate mean Phred scores for my sequencing data, but i feel not very comfortable about it. There are actually two ways of calculating. (I just use an existing sample) giving: 3 reads ...
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2 votes
1 answer
49 views

Excess average estimated identical-by-descent in genotype data

(Cross-post with Biostars: https://www.biostars.org/p/470788/) We have some genotype data that we are putting through quality control in PLINK 1.9. As part of this QC, we have limited the data to ...
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  • 133
1 vote
1 answer
51 views

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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1 vote
1 answer
145 views

Decontaminating RNA seqs for de novo transcriptome assembly and annotation of novel eukaryotes

I have raw paired-end RNA-seq reads for two novel eukaryotic species. Some background: the reads represent a copepod (arthropod) species each. The mRNA for each read set was obtained by extracting ...
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  • 111
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1 answer
146 views

Filtering raw sequencing reads

I have to filter the raw sequencing reads based on the following criteria: Remove reads containing adapters Remove reads containing N > 10% (N represents base that could not be determined) Remove ...
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0 votes
1 answer
313 views

What samples can be used for a Mutect2 Panel of Normals

I'm working on calling somatic variants from solid tissue tumors. I have one normal and one tumor sample for each patient. I plan to use Mutect2 to call somatic variants after preprocessing the data ...
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2 votes
2 answers
584 views

Importance of Proper Pairs vs Aligned Reads for RNASeq data

I have stranded, paired end RNASeq reads that I have aligned using STAR. I plan do conduct a differential expression analysis with DESeq2. After running quality control checks, a good portion of my ...
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3 votes
1 answer
96 views

Tool for calibrating base quality scores?

A given sequencing machine assigns a 'certainty' to each base call in the form of a per-base quality score (FASTQ format). I have reads from several machines aligned to the corresponding reference (...
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2 votes
1 answer
44 views

Metagenomic shotgun data with internal control

For 24 human stool samples I have metagenomic shotgun reads from an Illumina platform. The reads were filtered and mapped against a bacterial species library and specific maps to species were kept and ...
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2 votes
1 answer
291 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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1 vote
1 answer
172 views

Subset a multisample VCF file

I need some help with GATK. I have a multisample vcf file containing 18125 samples. After performing PCA I have around 303 samples as outliers and I wanted to remove these samples from my multisample ...
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4 votes
1 answer
142 views

Remove variants that do not map to human genome

[This question was also asked on Biostars] I received an hg38 VCF file that's had variants imputed with 1000 genomes. I've encountered some issues with the VCF; REF alleles that do not align to a ...
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6 votes
1 answer
38 views

Help with 1D^2 library shearing

I've done my 1st trial of 1D^2 whole genome sequencing using LSK-SQK308 kit with R9.5 flowcell. Without doing library fragmentation, I encountered the unexpected library shearing during sequencing. ...
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1 vote
0 answers
68 views

"Bad substitution" error while trimming adapters [closed]

I have this script to trim adaptors from my fastq sequencing files for PAIR in $(fastq | sed 's/_R.//g' | uniq) I am gitting this error: bad substitution Could ...
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3 votes
3 answers
267 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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3 votes
1 answer
3k views

Significance and timing of "mux scans"

I'm using MinIONQC to do quality control on some ONT data. The software plots several read characteristics over time (hours passed during the sequencing process). These plots contain several vertical ...
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7 votes
4 answers
6k views

Bash scripting FastQC for multiple fastq files in multiple directories

I am completely new to bioinformatics so I'm looking to learn how to do this. I have multiple directories with fastq files: E.g; 10 Directories with each time series, each with Treatment and control ...
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8 votes
3 answers
1k views

Why does cutadapt remove low quality bases from the ends of reads only?

I use cutadapt to remove low quality bases from my Illumina reads. The algorithm only removes low quality bases from the end until it reaches a good quality base. If there is a bad quality base beyond ...
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1 vote
1 answer
208 views

Optimal design for an RNA-seq experiment with ERCC RNA Spike-In Control Mixes

I am planning an experiment on gene expression. I have 6 conditions and 3 replicates in each condition. I would like to use ERCC spike-in mixes for quality check: lower limit of detection, dynamic ...
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2 votes
1 answer
113 views

How hard is it to clean and QC gene expression microarray data?

I am a PhD student working on developing ideas for my dissertation papers. One of my planned papers will be working with some (human) gene expression data. I have had a class on working with gene ...
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  • 195
8 votes
2 answers
7k views

Are mitochondrial genes to exclude in scRNA-seq such as ribosomal genes?

In this answer, it is stated that ribosomal genes should be excluded prior to normalization in scRNA-seq as contaminants. Do mitochondrial genes have to be excluded as well? I plotted the top 50 ...
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0 votes
1 answer
192 views

What is the --rev_comp_mapping_barcode parameter in the QIIME1 script extract_barcodes.py?

I'm using the QIIME1 scripts extract_barcodes.py to extract barcodes from a dual-indexed MiSeq library, and then demultiplex my reads using split_libraries_fastq.py. I have been having trouble ...
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12 votes
2 answers
3k views

determining doublets in single-cell RNA-seq

Doublets are a known problem with scRNA-seq experiments, where 2 or more cells are sometimes captured instead. To determine their presence, there are studies that mix multiple species (such as human ...
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  • 2,099
6 votes
2 answers
5k views

What are doublets in single cell RNA-seq data?

I am reading The Tabula Muris Consortium et al. (pp). In some organs, cells with more than 2 million reads were also excluded as a conservative measure to avoid doublets. How exactly is a “doublet”...
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4 votes
1 answer
229 views

Where can I get the population allele frequency vcf file?

I want to use GATK to estimate cross-sample contamination for Whole Genome Sequencing data. The specific tool is ContEst and it is run with: ...
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4 votes
2 answers
437 views

Error rate setting in Canu error correction

I want to use Canu to correct my nanopore long read (version: MinION R9.5), but I am not quite sure how to set the correctErrorRate. Should I follow the Canu manual (Nanopore R7 2D and Nanopore R9 1D ...
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  • 41
6 votes
3 answers
4k views

Removing PCR duplicates in RNA-seq Analysis

After reading some of the forum posts in Biostar and SeqAnswers I find it very confusing whether to filter out the duplicate reads from aligned files or not. As far I understand it's very difficult to ...
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5 votes
5 answers
584 views

QC measures for NGS sequencing

What are good means for performing quality control (QC) or NGS reads? I'm aware of: FastQC NGS Screen Kraken (e.g., for screening against contaminants) What are other popular means for such QC?
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