Hi Md Asaduzzaman ,

For small RNA, data requirement is low when compared to the total output of Hiseq X.

Hiseq X is capable of producing approximately 1 Tb of data from a single run.

So when loading small RNA samples with high data requirement samples, there are chances that the small RNA samples create clustering issues.

This will affect your data quality.

You can consider running your libraries in platform with lesser data output or along with samples of same data requirement.

Hi, Shan gave you a great answer. HiSeq X is a great instrument but does not have the same flexibility as other illumina very high-throughput sequencers (such as the Nova Seq series). I think this is why it is discontinued now and Illumina stopped selling them and sugges using the NextSeq or NovaSeq series.

Also, even RNA (transcriptome) sequencing should not be done of HiSeq X.

So it is definitely possible that using that instrument may cause a problem, we always sequenced or small RNA data (processed with the TruSeq Small RNA Library Preparation Kits by Illumina or the NEBNext Small RNA Library prep) on the "good old" HiSeq 2000/2500 or, recently, on the NextSeq 550. Never had any trouble with the reads.

Though the preparation of the libraries plays a major role in the quality of sequencing, using HiSeq X Definitely affected the results. It's not the ideal platform for small RNA sequencing. I would suggest using the MiSeq, MiniSeq or even the iSeq 100 for that purpose.

Good luck!