Fastq with 2 presence of adapter sequences

Question

[Armando]

hi all, I have a question: I have several samples with quiagen quiaseq miRNA library UDI kit, sequenced on Illumina Novaseq 6000 (1x76bp). When I check my quality fastq file, I obtained 2 adapter sequences shown in plot. (see the figure). Why? Can you help me?

Answer 1

[GenoMax]

Many miRNA libraries work by directly attaching an adapter to the miRNA followed by making Illumina libraries from the construct so they can be sequenced. So the reads that contain the kit specific miRNA adapter should be the real miRNA. Once that adapter ends you will sequence into the Illumina library adapter present after the miRNA adapter.

From Kit page:

In an unbiased reaction, adapters are ligated sequentially to the 3’ and 5’ ends of miRNAs. Subsequently, universal cDNA synthesis with UMI assignment, cDNA cleanup, library amplification and library cleanup are performed.

[Armando]

Thank you for the information. What about trimming? Is it necessary to remove both adapters? Or just the 3' NGS adapter from the Qiagen kit?

[GenoMax]

Yes you will need to remove the adapters. Since the Illumina adapter follows the smRNA one it should be removed automatically. Follow the analysis directions included with the kit.