PCR-cDNA PCB 109 Kit : Too many unclassified reads

[eKariuki-sleepy]

Hi, I have been experiencing large numbers of unclassified reads esp. with the pHMM backend. With the edlib backend, I get more full-length transcripts, but still the no. of unclassified is still significantly high. I sequenced using the PCB109 kit that multiplexes 12 samples. But it has VNP and SSPprimers that I assume are the same. How can I rescue more reads since my samples are multiplexed and I am only interested in unmapped reads in the long run? Going with these reads as recommended by pychopper, I might end up with very little data to work with since the coverage greatly reduces with multiplexing. Here is the HTML to my sequencing summary, and one of the pychopper reports. Thanks

[Ingway]

I used DCS109 without multiplex and my results are similar to yours, I got 32% primers found and 65% unusable. Does that meam that 65% of my reads are missing primers? I actually miss a better explanation of the outputs in the readme...

[damisiak]

Hi,

I have the same problem and wonder if it could be a problem by using the barcoded kit PCB109 instead of the kit without barcodes (PCS109)?

Any suggestions for that?

Cheers, Danny