Maybe a silly question for expert bioinformaticians, but I cannot figure out how to make stacks (v. 1.39) to recognize my combinatorial barcode file (3 columns file: barcode 5bp, index 6bp and sample name). Every time I run the command I receive an error message saying "Too many columns (3) specified in '/home/carol/RADseq/Barcodes/all_index-barcode' for single-end barcodes on line 1." Can someone help me to figure put the problem?
The command I have used is: ./process_radtags -i fastq -P -1 /home/carol/RADseq/Lane1_NonDemultiplexed/Lane1_read1.fastq -2 /home/carol/RADseq/Lane1_NonDemultiplexed/Lane1_read2.fastq -b /home/carol/RADseq/Barcodes/all_index-barcode —-index_inline —-barcode_dist_2 —-retain_header -o /home/carol/RADseq/Demultiplexed_Sequences/Demult_all_2ndtry -e pstI -D
Hi Carol !
You might have already found an answer to your question but just in case, maybe try this following command :
process_radtags -i fastq -P -p /home/carol/RADseq/(New directory name containing your raw data R1 and R2) -b /home/carol/RADseq/Barcodes/all_index-barcode —-index_inline —-barcode_dist_2 —-retain_header -o /home/carol/RADseq/Demultiplexed_Sequences/Demult_all_2ndtry -e pstI -D
Otherwise maybe try to switch -index_inline up to inline_index ; according to which of your barcodes is inline or in Fastq header. If your single end barcode is actually inline with the sequence, this could explain why Stacks is confused by a barcodes file showing more than one index for single end :), hence the error message it is returning you !
I am discovering Stacks as well so I could be totally mistaken, I would be happy to be corrected if I am wrong :)
Have a very nice day !
Maybe it is just the format of the barcodeFile.txt
Try save it with the separators indicated in STACKS manuals (e.g, windows text, tab-separated, etc.)
In a Unix environment it sometimes help using the strings command:
strings File.txt > File.unix.txt
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