remove gaps and extract 8bp

[JingGuo1997]

hi，scan-seq2 developer：
The single-cell third-generation transcriptome sequencing that you have developed is extremely exciting. When I replicate your data I have some doubts.
1、the code，My understanding is to remove reads whose length is less than 108bp, but I don't know where remove gaps is embodied in coding and why remove gaps should be removed.

########read length < 100 and remove gaps

seqkit seq -m 108 -g ${cell}_full_length.fastq > ${cell}_full_length_filtered.fastq
rm -f ${cell}_full_length.fastq
2、the code，removeing extract 8bp will truncate ploy A by 8bp in reads where umi has been removed. Why do you want to do this?

####remove extra 8 bp

cutadapt -u -8 -o ${cell}_full_length_filtered.fastq ${cell}_full_length_filtered.extract.fastq
rm -f ${cell}_full_length_filtered.extract.fastq

Sincerely look forward to your reply！

[liuzhenyu-yyy]

Hi Jing,

Glad to see that you're interested in our work. The gaps in fastq sequences are removed with seqkit:

seqkit seq -m 108 -g ${cell}_full_length.fastq > ${cell}_full_length_filtered.fastq

where -m specifies minimum read length and -g means remove gaps letters. Check more on the Usage and Examples page of seqkit. Technically, remove gaps improves quality of ONT reads mapping. In our pipeline it's just a habitual behavior and we had not test how much this could improve the performance of the pipeline,

An extra 8-bp is removed to deal with unexpected insertions or base errors caused by Nanopore sequencing and guarantee the ploy-A trimming step to work properly, Since ploy-A length was not included in our analysis, we figure would be ok to remove a few based in the end of ploy-A. This is not mandatory and should be adjusted according to you data.

Please let me know if you have any further questions.

[JingGuo1997]

Hi Zhenyu,
Thank you for your prompt response; your reply has been very helpful to me！