Small RNAseq data with untrimmed reads left after trimming process

Question

[MAPK]

Hi All, I have a general question about small RNAseq read size after trimming. I was able to trim my samples (sequenced with 50 bases long reads) with most of the reads resulting in 18-30 bases in length (which is expected of small RNA seq) after trimming. However, there are some reads that are still 50 bases long and their 3' region do not match with adapter sequence. Could it be possible for those reads to be pre-trimmed or come without 3' adapter justifying their length of 50 bases even after trimming? I was curious whether some of the reads could get sequenced without 3' adapter sequences. I would appreciate your expertise and clarification on this.

[GenoMax]

With small RNA seq if you are not able to identify the right adapter in your reads then it is likely not worth your while to try and figure out what those sequences are. Illumina sequencing is surprisingly sensitive. Many a times samples that seem to have little quantifiable DNA still produce sequence.

[MAPK]

As always, appreciate you answering my questions.

Answer 1

[h.mon]

Could it be possible for those reads to be pre-trimmed or come without 3' adapter justifying their length of 50 bases

It is highly unlikely to have some reads trimmed and some untrimmed in the same file (unless someone merged different files), so I don't think they were pre-trimmed. On the other hand, it is indeed possible some longer reads passed the size selection step during library preparation.

Do you have a reference genome available? If yes, map those "longer" reads to the reference genome and check if they match it over the entire sequence. Then you will be at peace about their origin and quality.

[MAPK]

Thanks, I checked those reads and there is 100% match with the genome. Any explanation for that?

[Shyam]

Where do these reads hit the genome? rRNA/tRNA? If the library prep is not good at the size fractionation you may get the fragmented rRNA/tRNA or other RNA classes might contaminate the library. If there is no 3' adapter in the reads, the insert might be longer than the 50 bp. You can do a BLAST at RNA Central.

[GenoMax]

If those reads did not follow the expected output structure based on the kit it is possible that they could be contamination of some sort. Not small RNA you are interested in.