How long should the AAA... sequence be at the end of read, so that fastqc and multiqc consider it as poly- tail

Question

[poecile.pal]

Good evening,

The multiqc report shows the presence of poly- tails in my samples after DNA (!) sequencing even after cleaning with fastp and cutadapt with appropriate settings. I think these are sequencing artifacts and I want to find reads with such "polyA-tails" in fastq.gz files.

Could you help me, please: how long should the sequence AAA... at the end ot read be so that fastqc and then multiqc consider it as a polyA-tail? This is necessary for search.

Thank you in advance, Poecile

P.S. By the way, these polyA-tails appear on adapter charts only after combining samples using multiqc, they aren't exist on fastqc charts for individual samples.

[GenoMax]

how long should the sequence AAA... at the end ot read be

If this is DNA sequence then there should be no poly-A's in the sense of poly-A tails.

I want to find reads with such "polyA-tails" in fastq.gz files.

Use bbduk.sh in filter mode with literal=AAAAAA (adjust the length as needed). If you need the poly-A's to be only in 3'-end of the reads then add restrictright=NN parameter.

the way, these polyA-tails appear on adapter charts only after combining samples using multiqc, they aren't exist on fastqc charts for individual samples.

Please show an example plot/table.

Answer 1

[Istvan Albert]

Don't trim polyA tails unless to problem seems to be acute

most aligners can align just fine, soft clipping the trailing As if those were present

long runs of As can naturally occur in transcriptomes and you run the risk of altering real data

Poly-A trimming - is it necessary?

Answer 2

[Jeremy Leipzig]

At least 12 A's (AAAAAAAAAAAA)

See https://github.com/s-andrews/FastQC/blob/d4f2e91d67b765867b59cde3c6a9a6c7de34ae37/Configuration/adapter_list.txt#L26

[Jeremy Leipzig]

Note that if the DNA fragment is shorter than the read length, as can happen with miRNA sequencing, Illumina will report the empty trailing space as a string of low quality A's

[GenoMax]

For this to happen with DNAseq you either will need very short inserts or some odd number of cycles in just one direction.

Even with miRNA you are not going to run out of sequencing adapter unless you sequence 100+ cycles in one direction.