barcode issues with proces_radtags

[Nathan Jones]

Hi all,

I am a new user to stacks and ddRADseq data processing.  I have a lot of experience with metagenomics but newb at this.  I am trying to preprocess (filter and demultiplex) two pools of paired end illumina data but process_radtags is throwing out all my sequences due to ambiguous barcode errors.  Any help is greatly appreciated.

Stacks version 1.44 My command is as follows.

process_radtags -P -p ./raw_pool_1 -b ./barcodes/pool_1_barcodes_v2.txt -o ./samples_pool_1/ \ -c -q -r --inline_index --renz_1 nlaIII --renz_2 mluCI \ -i fastq

My sequence file headers look like this

@K00179:78:HJ2KFBBXX:6:1101:1377:1209 1:N:0:GCCGCG+CGCGGC

My barcodes are present inline in the R1 sequence only my barcode file looks like this. 

GCATG

AACCA

CGATC

TCGAT

TGCAT

CAACC

GGTTG

AAGGA

AGCTA

ACACA

AATTA

ACGGT

ACTGG

ACTTC

ATACG

ATGAG

ATTAC

CATAT

CGAAT

CGGCT

CGGTA

CGTAC

CGTCG

CTGAT

the log file from process_radtags looks like this.

ss_radtags -P -p ./raw_pool_1 -b ./barcodes/pool_1_barcodes_v2.txt -o ./samples_pool_1/ -q -r --inline_index --renz_1 nlaIII --renz_2 mluCI  -c  -i fastq

process_radtags version 1.44 executed 2017-04-28 11:09:44

File Retained Reads Low Quality Ambiguous Barcodes Ambiguous RAD-Tag Total

17090Pool1_S9_L006_R1_001.fastq 0 0 51362358 0 51362358

17090Pool1_S9_L007_R1_001.fastq 0 0 51187232 0 51187232

Total Sequences 102549590

Ambiguous Barcodes 102549590

Low Quality 0

Ambiguous RAD-Tag 0

Retained Reads 0

Barcode Filename Total No RadTag Low Quality Retained

GCATG-AACCA CGATC 0 0 0 0

Sequences not recorded

Barcode Total

AATTA-CGCGG 9304968

ACACA-CGCGG 9264464

AGCTA-CGCGG 8649976

CAACC-CGCGG 7674144

AAGGA-CGCGG 7480068

GCATG-CGCGG 7479678

GGTTG-CGCGG 6973130

AACCA-CGCGG 6182070

[Nicolas Rochette]

Hi Nathan,

By indicating --inline_index you are specifying that your reads have barcodes both inline and in the adapters. If I understood you correctly what you want is --inline_null

Best,
Nicolas

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[Nathan Jones]

Thanks Nicolas, I tried changing that command and I think I am also having a problem with my bar code file.  The initial output from the previous failed run when I pass the command is that process_radtags only sees 1 barcode (GCATG-AACCA) GCATG is the first barcode in the file but I don't understand where -AACCA is coming from or why it doesn't see the other bar codes in the file.... 

When I tried this 
process_radtags -P -p ./raw_pool_1 -b ./barcodes/pool_1_barcodes_v2.txt -o ./samples_pool_1/ \ -c -q -r --inline_null --renz_1 nlaIII --renz_2 mluCI \ -i fastq

process_radtags begins the analysis but just quits after searching for inline barcodes....

how do you prepare your barcode files? I had them in excel and saved as a tab delimeted text file. Was this the wrong approach?

Cheers,

Nathan

[Nicolas Rochette]

Hi Nathan,

Could you post the log of the command that uses --inline_null? Preparing the file as you described works, but could you also show the result of

cat pool_1_barcodes_v2.txt | od -c | head

(n.b. Windows's '\r' character is not a problem.)

Nicolas

Nathan Jones wrote on 04/28/2017 01:44 PM:

[Nathan Jones]

Hi Nicolas,

The command with --inline_null I was passing didn't give any output other than to stdout but I think i've found the problem.  The barcode file had weird binary characters in it that were messing with the basic linux interaction.  I could open the file and view it in a text editor but using the more and head commands it would only show the first two lines.  For some reason my excel was adding non standard characters to the tab delimited output barcode file...  

It seems to work now.  Thanks so much for the help! I'm sure I'll have more questions when I get to actually analyzing some data and not just cleaning up! ;)

Cheers,

Nathan