Read length of RNA-seq from HiSeq 2500

Question

[boymin2020]

Hi,

Recently, I received some RNA-seq datasets from our co-operators. I have checked the qualities with FASTQC tool. The distribution of read length just includes 100bp. I want to know what is the sequencing range of fragments? Do the datasets have reads with <100 bp reads. If so, why they have not been shown here?

Thanks

[mastal511]

Illumina reads are all the same length, unless someone has trimmed them to remove adapters, low quality bases, etc., which is sometimes done by the sequencing center.

[boymin2020]

Thanks for the reply. Based on what you said, the fragments whose length are less than the specific sequencing length (e.g., 100bp) are not sequenced?

Answer 1

[GenoMax]

If the sequencing length is longer than length of fragment then the read will show read-through into Illumina adapter on 3'-end. You would still have a 100 bp read(s) until action is taken to scan/trim the extraneous sequence which will then result in a final read length of < 100 bp.

[boymin2020]

Thank for the reply. Could you also tell me how I see the read-though in illumina adapter?

[GenoMax]

If you scan/trim your data (I recommend bbduk.sh from BBMap suite), you will be able to see where the adapter sequences are. You could trim the data and then look at the corresponding original sequences in reads that get trimmed. Alternatively use the masking mode (kmask=N) to convert adapter sequences to N's, leaving the read length intact.

[boymin2020]

@genomax, thanks for introducing BBMap suite. But it seems I should know the adapter sequence before trim.

[GenoMax]

Not necessarily. If you are using one of the standard Illumina kits then the sequences necessary for scanning and trimming are included in a file (adapters.fa) in the resources directory of BBMap software distribution.