absence of overlapping nucleotides

[King_of_thenorth]

Hello NGS community. I have a question that I'd greatly appreciate your ideas on. We recently conducted an RNAseq analysis for a samples with a specific focus on small RNA to long non-coding RNA. To achieve this, we employed the P1 2x300 cycle sequencing approach to cover the entire range of RNAs, from approximately 20bp to 700bp because the Tapestation electropherogram showed peak from 175 to 700 bp. However, we have observed a decrease in run quality, and a significant portion of our reads had no overlapping sequences between Read 1 and Read 2.

Can someone explain to me why we are experiencing poor run quality and the absence of overlapping nucleotides?
Thank you!

[GenomicSeq]

King_of_thenorth to give us a little more background to help you, I listed a series of questions below. This will give us a more detailed understanding of what you did because otherwise there are a lot of reasons you could have had poor quality.

- What library prep did you use to prepare these samples?
- Have you ever used this prep before, and if so, what were the results?
- Can you share your Tapestation results? I was wondering if that would show anything.
- Did you perform a final cleanup on the samples?
- My final question is, what was the quality of the RNA before you ran it? I wasn't sure if there was any degradation.

But if you could answer at least some of these, then it would give us more information to help.

[King_of_thenorth]

Thank you for your questions GenomicSeq. I used Takarabio SMARTer® Stranded Total RNA-Seq Kit v3 to prepare the samples
This was my first time using the kit
-Although, the kit has been used in the lab for quite sometimes with no issues, except this was our first sequencing.
-Yes, tapestation data added here
Yes, according to the kit, there is clean up every after each PCR cycle, so yes I performed a final clean up
-I did not run the RNA tapestation before the library prep, but I used the QIAGEN RNeasy Micro Kit for RNA isolation from the and quantified them with the Ribogreen Assay to get the concentration. Those two kits have also been used for quite sometimes with no issues
-I assumed that I wouldn't have any issue with RNA quality as long as my cDNA libraries look good on tapestation
Thank you very much and I am looking forward to learning more from you.