Demultiplexing does not recognize 3' barcode

[tiagobrc]

Hi, Thanks for this fantastic tool!

I have been testing this tool on our pipelines and got into trouble when trying to demultiplex the 3' barcode on the second read pair.

The 5' demultiplexing works as a charm. So no problems regarding that.

I have a second-read pair that used a very similar barcode system.

Here is what my barcode.csv looks like.

I have a few questions that could not find in the manual or it wasn't clear enough.

1. Will Ultraplex automatically reverse complement my 3'bc for detection?
2. I actually have 96 barcodes combined with 16 sets ( these let me multiplex 16 different plates). Is there an easier way of creating the barcode.csv file? Instead of adding all combinations separated by a comma?

NNCGCTCAGTTCGG,NNCTGACCGGCA:P01A01
NNGCAGAGATGCAC,NNCTGACCGGCA:P01A02
NNGCAGAGACTCAG,NNCTGACCGGCA:P01A03
NNGCAGAGAGGAAT,NNCTGACCGGCA:P01A04
NNGCAGAGACGAGG,NNCTGACCGGCA:P01A05
NNGCAGAGAAGGAG,NNCTGACCGGCA:P01A06
NNGCAGAGATGTTG,NNCTGACCGGCA:P01A07
NNTGCCTTGATCGG,NNCTGACCGGCA:P01A08

Read1

CA CGCTCAGTTCGG Adapter1 - Rest of DNA
AC GCAGAGATGCAC Adapter1 - Rest of DNA
GC GCAGAGAGGAAT Adapter1 - Rest of DNA
TA GCAGAGAAGGAG Adapter1 - Rest of DNA
GG GCAGAGAAGGAG Adapter1 - Rest of DNA
TG TGCCTTGATCGG Adapter1 - Rest of DNA

Read2

CA CTGACCGGCA Adapter2 - Rest of DNA
AC CTGACCGGCA Adapter2 - Rest of DNA
GC CTGACCGGCA Adapter2 - Rest of DNA
TA CTGACCGGCA Adapter2 - Rest of DNA
GG CTGACCGGCA Adapter2 - Rest of DNA
TG CTGACCGGCA Adapter2 - Rest of DNA

[simonpfisterer]

Have you been able to solve this issue? I have the same problem that 3' barcodes are not detected even though I believe that they are correctly specified

[Delayed-Gitification]

Hi, please send me a sample of your fastq and your barcodes csv

[simonpfisterer]

Hi thanks for offering this! Hope it is a simple issue with the 3'barcode. Please excuse the labelling of the barcode file, but I think I went through too many versions.
As shown in post below. I solved it myself, thanks for offering your help!!

[simonpfisterer]

OK, I solved it myself. It is the min_trim=3 default setting. It removed the last three nucleotides of the barcode and therefore it could not be matched. I will remove my data files from above.

[tiagobrc]

I will try this suggestion.