Shifu Chen comments

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                                            Shifu Chen

Duplication rate differs up to 30% from that of Fastqc for single end reads

Fastp duplication rate was calculated on the original data.

fastp version 0.23.0 and above failed to process interleaved Fastq files

Will fix it soon.

fastp version 0.23.0 and above failed to process interleaved Fastq files

fixed, please update to v0.23.2

Insert size peak 35 bp

How long you sequenced? Have you checked the template length peaks with QC devices like Agilent 4200?

Insert size peak 35 bp

Would you please upload the HTML report?

Insert size peak 35 bp

From the reports. OV080516 DNA fragments are too short, and OV982816 DNA fragments are too long (so no overlap). Furthermore, both them have a lot of primer dimers.

Insert size peak 35 bp

In OV982816, 99.8% reads are with unknown template length, which means the read pairs are not overlapped, which means the template length is > 2*250. So the DNA fragments are...

Insert size peak 35 bp

Insert size can be much much higher than read length. Actually you can evaluate the insert size again using the alignment file, which can be more accurate.

5' Adapter Trimming

Did you mean to remove some pattern sequence inside the reads?

Option for overrepresented sequence

It's not reasonable to trim/filter overrepresented sequence. If you want to remove deduplicated reads, use --dedup