Invalid barcode on line 1 (process_radtags)

[Dongmin Kim]

Hi I am a new user to stacks (latest version).. I am running through bash (Ubuntu) and have no idea what to do... 

Can anyone explain how barcodes and my raw data can work together?.. I need to step by step tutorial I guess or just need to understand how these all work.

So here is the barcodes text file that I got from my professor that he used for his initial analysis 

ACTAGG ATCACG P18

AGCATT ATCACG P10

AGTAAG ATCACG NW15-8

ATAACC ATCACG Bee15

ATGTCC ATCACG Bee2

CAGAGT ATCACG P17

CATCTC ATCACG Bee16

AACGGT ATCACG P8

CCCATA CGATGT NE21

CGAAAC CGATGT CR16

CGAATG CGATGT E08

GACCAA CGATGT J18

GACGTT CGATGT J03

GCAGAA CGATGT Bee17

GGGATA CGATGT NE1

GTCTAT CGATGT Bee10

ACTAGG TTAGGC CR15

AGCATT TTAGGC Bee4

AGTAAG TTAGGC CR12

ATAACC TTAGGC E03

ATGTCC TTAGGC NN08

CAGAGT TTAGGC J02

CATCTC TTAGGC Bee1

AACGGT TTAGGC NE18

CCCATA TGACCA NW15-3

CGAAAC TGACCA P11

CGAATG TGACCA Bee6

GACCAA TGACCA P7

GACGTT TGACCA NW15-10

GCAGAA TGACCA NW15-7

GGGATA TGACCA NW15-15

GTCTAT TGACCA P13

ACTAGG ACAGTG NW15-14

AGCATT ACAGTG NW15-11

AGTAAG ACAGTG H15-1

ATAACC ACAGTG Bee3

And here are the first few lines of the sample file that I am using to see how process_radtags works

@D00723:44:HJF35BCXX:1:1101:2574:2242 1:N:0:ATCACG

GACATAGGAACGGTTGCAGGGGGTTAGAGCTGTTGGACATTGTGGAACATTCTGTGGAGTAGAGGTGTGCACGCATGTTCTCCATGCTGACTGCAGGAGG

+

GGGGGGGIIIIIIIIIIIIGIIIIGIGIIIIIIIIIIIIIIIIIIIIIIIGIIIIIIIIIIIIIGIIGGGGGIIIIGGGGIGIIIGIIGGGIIIGGIAGI

@D00723:44:HJF35BCXX:1:1101:2843:2247 1:N:0:ATCACG

TGGCACACGTCTGAACTCCAGTCACATCACGATCTCGTATGCCGTCTTCTGCTTGAAAAAAAAAAAAAAACACCCAATCCGACCGACCTCAATCGCAAAA

+

AGAGAGIGGIIIGGIG<<GGG<GAGGGGGIGGAGGGIIIGIIIGGGGGGGAGAGGGGGIAAGGG<A....<.<.....<...<....<G..<.<..<A..

@D00723:44:HJF35BCXX:1:1101:2777:2247 1:N:0:ATCACG

GTCGATGAAGCATTTGCAGGTGATTGAGGCTCCAGACTGCCTTTGCCAGCAGACACAGGGACGACTGATGCAGAGGACTGGTCAAATTTGGTGGTGCAGA

+

GGGGGGIIIIIIIIGIIIGIIIGIIIGIGGGGIIIIIIGGIIGIIGIIIIIIIIGGIIIGIIIIIIIIIIIIGGIIGGGGGGIGIIIIIIIIGIIIIIII

@D00723:44:HJF35BCXX:1:1101:3200:2244 1:N:0:ATCACG

GGCACACGTCTGAACTCCAGTCACATCACGATCTCGTATGCCGTCTTCTGCTTGAAAAAAAAAACACAGATAAGTTCCACAATAGACAACACCAAATTTA

+

GGGGGIIIIIIIIIIGIIIIGIGIIIIIGGIIIIIIIIGIIIIGGGIIIIGIIIIIIIIIIIGG.<...<..<............<.........<....

when I used this command, ./process_radtags -p . -o ./samples/ -b ./tmp -e sbfI -r -c -q

It says that 

Processing single-end data.

Using Phred+33 encoding for quality scores.

Found 1 input file(s).

Searching for single-end, inlined barcodes.

Invalid barcode on line 1: '@D00723:44:HJF35BCXX:1:1101:2574:2242 1:N:0:ATCACG'

what should I do?

[Natalia Bayona Vásquez]

Dear Dongmin Kim,

Your barcodes seem to be the first part of the sequence and it is not present in the fastq head, then you need to specify --inline_null for single end or --inline_inline for paired end,for example: 

process_radtags -P -p . -b ./tmp -q -c -r --inline_inline  -o ./samples/

[Dongmin Kim]

Dear Natalia 

So that means I should edit my fastq file (tmp) and add barcodes as the first part of the sequenc? 

So for example, 

If my first line of the raw data looks like this 

@D00723:44:HJF35BCXX:1:1101:2574:2242 1:N:0:ATCACG

GACATAGGAACGGTTGCAGGGGGTTAGAGCTGTTGGACATTGTGGAACATTCTGTGGAGTAGAGGTGTGCACGCATGTTCTCCATGCTGACTGCAGGAGG

And barcode is 

ACTAGG

Should I add like this?

ACTAGG @D00723:44:HJF35BCXX:1:1101 2574:2242 1:N:0:ATCACG GACATAGGAACGGTTGCAGGGGGTTAGAGCTGTTGGACATTGTGGAACATTCTGTGGAGTAGAGGTGTGCACGCATGTTCTCCATGCTGACTGCAGGAGG

which part of barcode do I need to add in front of the first line? And do I need to do that to evert single raw fastq files that I have? 

[Julian Catchen]

Read some introductory papers:

https://www.nature.com/articles/nrg.2015.28
https://www.nature.com/articles/nrg3012
http://onlinelibrary.wiley.com/doi/10.1111/mec.12354/abstract

Read the manual:

http://catchenlab.life.illinois.edu/stacks/manual/

Read the Nature Protocol:

https://www.nature.com/articles/nprot.2017.123

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Julian M Catchen, Ph.D.
Assistant Professor
Department of Animal Biology
University of Illinois, Urbana-Champaign
--
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[Natalia Bayona Vásquez]

No, you don't need to edit any file. 

You can check in the webpage what are the options to specify barcodes and why I am suggesting you to use --inline_inline.