Only a small portion of my reads have Smart-seq adapters, why?

Question

[Youyy]

I am new to the SMART-seq technology, I followed the SMART-Seq® Stranded Kit User Manual by the Takara Bio USA, Inc. After I got my raw data for a sample, I followed the tuxedo pipeline to process the paired end data, FastQC -Cutadapt/Trim Galore - Tophat/HISAT2 - Cufflinks/Stringtie.

For the Cutadapt, I entered my SMART-seq adapters, For each of R1 and R2, I entered my 3' adapter, ATAGAGGC, and 5' adapter GAATTCGT. My quality cutoff is 20, and the minimum length is 20.

Then I got the report:

However, I only got a very small portion of my reads with SMART-seq adapters, read1 with adapters is 6.8%, read2 with adapter is 2.3%. What are the possible problems? Thank you so much.

Answer 1

[ATpoint]

There are no problems most likely, this is normal and expected. A good library prep makes the insert size (the actual cDNA part between the adapters) long enough so that standard read lengths do not sequence "into" the adapters as this is waste of resources. Insert size is not "fixed" though like "exactly" e.g. 200bp, it is somewhat Gaussian with a slight right-skewed distribution, so most fragments are longer than the read length, and only few actually are shorter so adapter content is being detected during sequencing for these. Nothing to worry about here most likely. Trim them and go on with alignment.

[Youyy]

Thank you so much. I don't understand the algorithm of the tools well, so I am not clearly how they exactly treat the reads.

[Youyy]

If I use Trim Galore, does it remove the SMART-seq Adapters? Because there is no option to specify SMART-seq adapters.