Can I pass a fastq file twice in trimmomatic?

Question

[Vitor1]

I would like to know if I can pass a fastq file in trimmomatic, and then pass again its results. Also, how can I be sure that a fastq file that I downloaded from public sources has already been trimmed for quality?

I got some SRA fastq files that seems that they have already been trimmed, since their reads are not the same lengh. But when i look at them in the Fastqc, it redflags a significant number of reports (as for example GC contents and overrepresented sequences). When I pass it again in trimmomatic, some of the reports in Fastqc get a better. So my doubt is if it's ok to do this, pass them twice in the trimmomatic. In the comments are some examples of what I got.

Thanks

[cpad0112]

it can be done. But it would help us understand if you could tell us why two pass is necessary for your work.

[Vitor1]

The thing is that I got some SRA fastq files that seems that they have already been trimmed, since their reads are not the same lengh. But when i look at them in the Fastqc, it redflags a significant number of reports (as for example GC contents and overrepresented sequences). When I pass it again in trimmomatic, some of the reports in Fastqc get a better. So my doubt is if it's ok to do this, pass them twice in the trimmomatic. Here goes some images that explain what I got. (The Filename is after passing is with a 6 in the place of 5, It was a typo)

[cpad0112]

There are no restrictions on number of times in filtering reads.

Answer 1

[Istvan Albert]

If it is Illumina data and the reads are not the same lengths then it has been trimmed.

You can pass files as many times as you want, now what happens is not always obvious, normally one would expect that nothing will change second times around, but in reality, algorithms may work in unexpected ways, have various initial conditions, thus the second time you might still get changes (I don't how Trimmomatic works here, I am just pointing out that it could happen even if seems counterintuitive)