all reads invalid

[Yang990-sys]

hello,

I'm using tailfindr to find polyA/polyT tails for cDNA ，and my kit is SQK-PCS111, but all reads are invalid,

I have re-basecalled my fast5 file with guppy :

guppy_basecaller \ --config dna_r9.4.1_450bps_flipflop.cfg \ --input $INPUT \ --save_path $OUTPUT \ --recursive \ --fast5_out \ --hp_correct 1 \ --disable_pings 1 \ --enable_trimming 0

and find tails with:

find_tails(fast5_dir = './test/', save_dir = './', csv_filename = 'cdna_tails2.csv', num_cores = 20, dna_datatype = 'pcr-dna', basecall_group ='Basecall_1D_001', save_plots = F)

Is this because primers are not specified ? Or other reasons? Thanks so much for your help !

[adnaniazi]

Hi, Please remove dna_datatype = 'pcr-dna', from your command, and try again. Hopefully it would work.

Best, Adnan

[Yang990-sys]

Thanks so much for your help. when dna_datatype = 'pcr-dna' is removed, I get 2715 valid reads in 4000 reads. Within these invalid reads, 895 reads still seems to be read_type:invalid ,345 reads is tail_is_valid:FALSE, and 45 reads is all blank. Is that a normal ratio?

🙏🙏

[adnaniazi]

Yes, the number seems normal to me in my experience.

Adnan

[Yang990-sys]

Thanks a lot for your help !