Questions tagged [quality-control]

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11
votes
2answers
2k 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 ...
8
votes
3answers
820 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 ...
7
votes
2answers
4k 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 ...
6
votes
2answers
2k 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”...
6
votes
3answers
3k 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 ...
5
votes
3answers
3k 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 ...
5
votes
0answers
29 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. ...
4
votes
5answers
504 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?
4
votes
1answer
197 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: ...
4
votes
2answers
331 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 ...
3
votes
3answers
149 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 ...
3
votes
1answer
732 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 ...
3
votes
1answer
39 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 (...
3
votes
1answer
110 views

Remove variants that do not map to human genome

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 reference genome, ALT alleles that do not ...
2
votes
2answers
74 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 ...
2
votes
1answer
89 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 ...
2
votes
1answer
93 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 ...
1
vote
1answer
88 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 ...
1
vote
1answer
139 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 ...
1
vote
1answer
60 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 ...
1
vote
1answer
33 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 ...
1
vote
0answers
43 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 ...
0
votes
1answer
43 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 ...
0
votes
1answer
86 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 ...
0
votes
1answer
129 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 ...
0
votes
0answers
9 views

Common QC parameters for SNP chip data in PLINK

How do you set QC measurements? Seems very subjective. Is there a right way to set: 1) individual call rate, 2) SNP call rate, 3) minor allele frequency (MAF), and 4) linkage disequilibrium (LD) in ...
0
votes
0answers
32 views

Quality checking benchmarks for clustering

I'm currently working with several RNAseq datasets from the ENCODE database. My approach to assessing the quality of the data, i.e, reproducibility is by automating the assessment of expression ...