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
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.
...
- 6,485
6
votes
Accepted
"Sequence Duplication Levels" module still fails after pre-processing Illumina data
To answer your direct question, there are a few reasons why there might be high levels of sequence duplication. From the FastQC help:
The underlying assumption of this module is of a diverse ...
- 5,060
6
votes
"Sequence Duplication Levels" module still fails after pre-processing Illumina data
FastQC assumes that all samples are for whole genome sequencing and will flag them as failed if they differ too much from that assumption. This will, for example, cause essentially all RNA-seq, ChIP-...
- 19.6k
6
votes
Accepted
FASTQC overrepresented sequences after trimming
As @AaronBerlin mentioned, you didn't remove reads that were completely trimmed. Next time use the --minimum-length option and set it to something reasonable, like ...
- 19.6k
5
votes
Accepted
Telling grep to treat N as [ATCG]
If you want to stick to grep, use a scripting language such as Perl to generate the regex programmatically. For example:
...
- 839
4
votes
Accepted
insert size pre and post trimming
It's not an unreasonable approach. rMATs is rather picky about its input, but it seems you've noticed that.
rMATs can't handle trimmed reads. It also can't handle soft-clipped reads. You might as well ...
- 19.6k
4
votes
How can I systematically detect unknown barcode/adapter sequences within a set of samples?
You mention that FastQC "fails to find the actual adapter sequences" - I guess you mean in the Adapter Sequence Contamination plot. However, the kmer and Sequence Content Plots are often useful even ...
- 291
4
votes
Accepted
How to trim adapter sequences from GSE65360 in order to map the reads?
First the Nextera adapters and the custom barcode adapters overlap each other
...
- 2,574
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
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 ...
- 912
3
votes
fastq file format unknown
This indeed looks like some sort of binary file. I have seen bunch of those when people renamed .fastq.gz to .fastq without ...
- 5,517
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 ...
- 5,517
3
votes
Accepted
What are the right parameters to trim a small RNA transcriptome with trimmomatic?
The command where you trim with adapters and by quality is perfectly fine. That FastQC isn't perfectly happy is expected. It's a tool made for whole genome sequencing QC, you SHOULD see a number of "...
- 19.6k
2
votes
Is there a safe catch-all adapter sequence for trimming?
Alternatively, I really like using bbduk which is part of the BBMap suite.
I've processed every nascent sequencing dataset that has been published, and found a lot of quirky errors with older ...
- 141
2
votes
Is there a safe catch-all adapter sequence for trimming?
You're best off just using fastp or Trim Galore!, both of which will determine the adapter sequence for you. Trim Galore! uses a ...
- 19.6k
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
How can I systematically detect unknown barcode/adapter sequences within a set of samples?
I'm not aware of any existing methods to do this, but here are a couple of ideas about how it might be done:
Canu has a method of adapter trimming which involves looking for the absence of overlap ...
- 13.8k
2
votes
Accepted
Using sickle for quality trimming
It'd be rare to run into Illumina encoded quality scores any more, I doubt there are machines running that still produce those. Everything produced at the moment uses Sanger encoded (phred + 33) ...
- 19.6k
2
votes
Accepted
Paired end reads with different read lengths
I find that the forward read length is 50 while the reverse reads length is 35. How is this possible?
Forward cycle length was longer on SOLiD than reverse cycle length; this is common for SOLiD data....
- 13.8k
2
votes
Accepted
Primer trimming-fasta files
The IUPAC ambiguity codes can be thought of as regular expression character classes. R matches any purine, so [AG], ...
- 9,662
2
votes
Accepted
Trim reads 1kb upstream of sequence
Here's one way using biopython. Note that the Seq object has a number of methods that act just like those of a Python string. One of these is the ...
- 3,059
1
vote
1
vote
How to run trimmomatic in HPC
Depending on the cluster management tool, you might have received e-mails when the "job" begins and ends. If so, you can check the "Exit status" of the job. For example, in the case of our HPC the ...
- 3,947
1
vote
Why does cutadapt remove low quality bases from the ends of reads only?
For Illumina (and 454) reads, the quality decreases with read length. It's not linear and is run/library-dependent. It has less to do with exhaustion of reagents and more to do with the strands on a ...
- 106
1
vote
Accepted
How can I systematically detect unknown barcode/adapter sequences within a set of samples?
The minion utility from the kraken/reaper toolkit may be helpful for this: http://wwwdev.ebi.ac.uk/enright-dev/kraken/reaper/src/reaper-latest/doc/minion.html
- 315
1
vote
How can I systematically detect unknown barcode/adapter sequences within a set of samples?
If you happen to know a sequence that should be highly abundant in the library, you can grep its beginning or end (with pattern match highlighting) and see if the same sequence systematically comes ...
- 3,100
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