12
votes
QC measures for NGS sequencing
MultiQC can merge all your different reports into a single one.
Which could be useful once you manage to know which QC tools to use.
- 129
10
votes
determining doublets in single-cell RNA-seq
Expected rates of doublets / duplets / multiplets
Fluidigm C1 doublet rate: around 1-5% depending on chip type used.
More information: Fluidigm white paper: Redesign of C1 Medium-Cell 96 IFCs ...
- 2,624
9
votes
Why does cutadapt remove low quality bases from the ends of reads only?
If you check the read QC statistics of an Illumina run in e.g. fastQC, you will see that at the end of the read the quality decreases. This is because of exhaustion of chemicals at the end of the run. ...
- 3,571
9
votes
Accepted
Removing PCR duplicates in RNA-seq Analysis
For normal RNA-seq PCR duplicates are normally kept in, but the duplication rate can be used as a quality control: The higher the duplication rate, the lower the quality. For expression analysis, it ...
- 3,271
7
votes
Accepted
Why does cutadapt remove low quality bases from the ends of reads only?
I am commenting on this part:
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 that, it is not trimmed.
...
- 6,445
7
votes
QC measures for NGS sequencing
We routinely run both FastQC and FastQ Screen on all of our raw sequencing reads. FastQ Screen is a tool for detecting cross-species contamination. MGA is another similar tool.
There are then lots of ...
- 291
7
votes
What are doublets in single cell RNA-seq data?
"Doublet" is commonly used to describe a droplet in droplet-based sequencing that has captured atleast 2 cells. 10x states their doublet rate to be 0.8% per 1000 cells:
There is a tradeoff between ...
- 71
7
votes
Accepted
What are doublets in single cell RNA-seq data?
Is doublet a set of cells sequenced as a single cell?
Yes. Depending on the method of single cell sequencing it may be more or less likely for groups of cells to be captured and barcoded with the ...
- 13.5k
6
votes
Accepted
Are mitochondrial genes to exclude in scRNA-seq such as ribosomal genes?
According to Ilicic et al. (2016), on upregulation of mtRNA in broken cells:
There is an extensive literature on the relationship between mtDNA, mitochondrially localized proteins, and cell death [...
- 1,783
6
votes
Significance and timing of "mux scans"
Yes, your understanding is largely correct. This originates from the situation that for each detector on a nanopore array there are 4 pores. I'll explain mux scans and groups, but this is outdated ...
- 1,314
5
votes
Bash scripting FastQC for multiple fastq files in multiple directories
multiqc kind of glazes over some important information, like the exact adapters and duplicated sequences in a library. If you plan to spend big $$ for sequencing a ...
- 3,141
5
votes
Accepted
Where can I get the population allele frequency vcf file?
On the GATK forum they've recommended the population stratified VCF file for this purpose.
- 19.5k
5
votes
QC measures for NGS sequencing
The quality control of ngs reads is heavily dependent on type of the project.
For genome assembly projects based on short reads, beside already covered checking quality of sequencing, you would like ...
- 5,497
5
votes
Accepted
What is the right way of calculating a Phred score by hand?
Not sure if my explanation is any good but let's try...
First, let's convince ourselves that converting phred to probability is the right thing to do (i.e. your Way 2).
Imagine a read of length 10000 ...
- 689
5
votes
PL and QUAL values on VCF file?
The VCF specification provides the definition for the QUAL field.
However, QUAL values are often capped by variant callers to a given value. I have seen 100 being used and according to this ...
- 750
4
votes
determining doublets in single-cell RNA-seq
We cannot assume that doublets will produce more UMIs
I would caution the assumption that all doublets will have twice the UMI levels of isolated single-cells. Many "doublets" could contain ...
- 873
4
votes
Tools for quality trimming at 5 prime?
You can have a look at cutadapt. It is capable of quality trimming for both ends as you can read here.
- 629
3
votes
Oxford Nanopore mapping Quality and sequencing error
Where i know with the phred score mapQ of 60 means 99.9999% of the bases are called correctly.
That's not at all what the MAPQ means, it means that the sequence is very likely to have originated from ...
- 19.5k
3
votes
Accepted
Subset a multisample VCF file
Based on the way you've called the command (using -xl-sn), I assume you are using GATK 4 and not GATK 3.
If this is the case, the documentation for the ...
- 375
3
votes
Tools for quality trimming at 5 prime?
I never spent too much time on choosing my trimming software, therefore I might have missed some jewels. I use trimmomatic when I need versatility and I am entirely sure that it trims reads on both ...
- 5,497
3
votes
Error rate setting in Canu error correction
It's generally a good idea to trust the "official" suggestions. You can also adjust the error rate based on coverage according to parameter reference:
For low coverage datasets (less than 30X), we ...
- 2,159
3
votes
Removing PCR duplicates in RNA-seq Analysis
Generally you should just leave them as is. One does remove/mark duplicates in DNA seq.
For further read check this Nature paper
- 51
3
votes
Accepted
Does cutadapt trim trailing N's first and then use max_n to filter reads?
Yes, it behaves just like you expect, where a read with >40% Ns but only at the ends will still be kept (after trimming).
Cutadapt runs in two stages, with read modification first and read ...
- 812
2
votes
QC measures for NGS sequencing
The kind of QC you do routinely depends on what your lab's focus is. We do a lot of low-quality, multiplexed DNA and RNA. If you routinely do fresh frozen whole genomes, your QC will be different.
...
- 540
2
votes
Accepted
Optimal design for an RNA-seq experiment with ERCC RNA Spike-In Control Mixes
I have 6 conditions and 3 replicates in each condition.
If including more biological replicates has a low experimental cost, you should be using 6 replicates per condition (and fewer reads per ...
- 13.5k
2
votes
Accepted
How hard is it to clean and QC gene expression microarray data?
I am not sure how much you know about bioinformatics already, can you use R? For a bioinformatician looking at QC for microarrays should not be a big deal, at least for me it would take maybe a day (...
- 3,571
2
votes
Are mitochondrial genes to exclude in scRNA-seq such as ribosomal genes?
Mitochondrial genes do not always have to be removed, though you may need to do so for a variety of technical reasons.
Mitochondrial reads are innately different from rRNA due to rRNA typically being ...
- 19.5k
2
votes
Tools for quality trimming at 5 prime?
Out of all of the major trimming tools available and widely used (trimmomatic, cutadapt/trimGalore (trimGalore is built on top of cutadapt), fastp), I actually instead prefer bbduk which is part of ...
- 141
2
votes
Bash scripting FastQC for multiple fastq files in multiple directories
This is the typical kind of job for snakemake.
Assuming your have one file per replicate named for instance T9/Infected/Rep1/Rep1.fastq.gz, you can prepare a file ...
- 3,090
2
votes
Importance of Proper Pairs vs Aligned Reads for RNASeq data
If you check the RSeQC docs you can get an explanation of how the inner distance stats work. By the look of your inner-distance MultiQC plot I guess that your sequencing is 2x100bp reads. This plot ...
- 291
Only top scored, non community-wiki answers of a minimum length are eligible