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Drosophila ultra low input RNA seq results - excessive clontech smart cds primers

Hey everyone! I got results from an ultra low input RNA seq experiment back. Some relevant information about the sequence approach:

1) Sequenced drosophila embryonic neurons post FACS sorting 2) Samples were trimmed by company 3) Low input library prep is proprietary but comparable to SMART-Seq v4 4) paired end sequencing

I used kallisto to pseudoaligned trimmed reads, then fastqc/multiqc for quality control.

A few observations from the fastqc/multiqc:

1) the second read of all samples are messed up - quality is low (see comparison below:)

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2) Upon closer inspection, it appears are though I have sequenced a ton of the clontech smart CDS primer:

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3) Lastly, here is a global view of the samples:

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I have no clue how to proceed forward with this analysis? Could this pseudolignment be partly to blame for the ~40-50% alignment? Should I use STAR instead? Lastly - would it be beneficial to trim the clontech primer sequences from the raw reads and proceed with Kallist/STAR -> downstream analysis?

Any help or thoughts would be greatly appreciated. Thanks!

drosophila low-input rna-seq
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updated by ATpoint ATpoint • written by barrbarrbinks • barrbarrbinks

Do you have adapter contamination popping up in the fastqc report? My guess for that R2 would be that those later cycles where quality is lower might be due to the sequencing hitting the polyT stretch in the fragment. What is the average insert size of these libraries (based on bioanalyzer or similar approaches)? If there is adapter content then I would trim that. STAR might just soft-clip it.

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written by ATpoint ATpoint

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