Stacks 2.0: problem with paired reads

[sd...@sanger.ac.uk]

Hi guys, 

We are running some tests using 2.0 using pairder-end ddRADseq data. denovo_map.pl completes perfectly when "--paired" is not used, ie. only R1 is used, however, It crashes when the "--paired" tag is used, and reports the following:

Error: Failed to find any matching paired-end reads in '/nfs/***/steve/denovo/FL2_0_Pre_06.2.fq.gz'.

Aborted.

The fastqs are directly out of process_radtags, and they look fine to me. Is it looking for something in particular with the read name? /1 /2 formatting?

Cheers, 

Steve

[sd...@sanger.ac.uk]

Solved...

Looks like process_radtags was adding a /1 /2 to the demultiplexed reads, which was in addition to the existing /1 /2. My bad.

I presume "--retain_header" would have made this a non issue from the start... ?

Cheers, 

Steve

[Pedro]

Hi

I have this problem also with stacks 2.0 beta7c...

The sequence header in the fq.gz already contains info about the paired reads as 1:N:0 or 2:N:0 at the end of the header.  Process_radtags is adding the respective /1 and /2 to the header. The problem is when using the tsv2bam: the header does not match between paired reads because of the 1:N and 2:N, so it fails to find matching reads... It does work if I replace the paired sequence headers with 1:N...

Cheers
Pedro

[Julian Catchen]

Hi Pedro,

The Stacks programs are designed to input the FASTQ files generated by
process_radtags. I would not use '--retain_header' for the data you will
push through the Stacks pipeline, so that it has the proper /1 and /2
designations.

julian

[Pedro]

Hi Julian

Thanks for your fast reply!

In fact I did not use the '--retain_header'. To be more clear, I have Illumina reads
that have, for example, this header:

@K00133:338:HK37MBBXX:7:1101:30594:1015 1:N:0

and for the paired:

@K00133:338:HK37MBBXX:7:1101:30594:1015 2:N:0

The process_radtags is adding the respective /1 and /2 at the end of the header (and also to the name of the file that I have to rename afterwards). The problem is when I have to run the tsv2bam. It fails to match the paired reads because the header does not match before the '/1' and '/2' due to the different '1:N:0' and '2:N:0'. Since this is the standard Illumina header for paired-end sequencing, shouldn't it be parsed instead of trying to find a full match?

I managed to resolve the issue by replacing all headers in the paired reads. Now it finds all matching sequences.

Cheers

[Nathan Jones]

Hi Pedro,

Any advice on a quick to replace all the headers in the paired end reads?  I am also having this issue with the /1 /2 on my illumina PE reads...

Nathan

[Julian Catchen]

Hi Nathan,

Can you send a sample of what your raw data look like and what they look
like after process_radtags is run?

Thanks,

julian

[Nathan Jones]

Hi Julian,

Sample of top of the fastq files after process radtags.

[Nathan Jones]

And here the top o the raw files from the sequencing facility.  

[Julian Catchen]

Hi Nathan,

Based on the files you sent me, the software is working as expected

Raw:

@K00179:78:HJ2KFBBXX:6:1101:1377:1209 1:N:0:GCCGCG+CGCGGC
@K00179:78:HJ2KFBBXX:6:1101:1925:1209 1:N:0:GCCGCG+CGCGGC
@K00179:78:HJ2KFBBXX:6:1101:2737:1209 1:N:0:GCCGCG+CGCGGC
@K00179:78:HJ2KFBBXX:6:1101:2899:1209 1:N:0:GCCGCG+CGCGGC
@K00179:78:HJ2KFBBXX:6:1101:3001:1209 1:N:0:GCCGCG+CGCGGC
@K00179:78:HJ2KFBBXX:6:1101:3244:1209 1:N:0:GCCGCG+CGCGGC

Processed:

@6_1101_8237_1244/1
@6_1101_11688_1244/1
@6_1101_25611_1279/1
@6_1101_22374_1297/1
@6_1101_23652_1297/1
@6_1101_28605_1297/1
@6_1101_11322_1314/1
@6_1101_19299_1314/1

And the pairs match up as expected:

@6_1101_8237_1244/2
@6_1101_11688_1244/2
@6_1101_25611_1279/2
@6_1101_22374_1297/2
@6_1101_23652_1297/2
@6_1101_28605_1297/2
@6_1101_11322_1314/2
@6_1101_19299_1314/2

What am I missing?

julian

[Julian Catchen]

You will want to read about the outputs for process_radtags:

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

By definition the remainder files do not have pairs, as the other read
was discarded due to quality, you should not try to push the remainders
through the pipeline.

Nathan Jones wrote:
> When the denovo_map.pl <http://denovo_map.pl> gets to the stage of
> tsv2bam I get this error in the log file.
>
> Paired-end reads files found, e.g. './samples_all_PE/060_01PI1.rem.2.fq'.
> Loading the catalog...
> Processing sample '060_01PI1.rem'...
> Processing sample '060_03PI1.rem'...
> Processing sample '060_02PI1.rem'...
> Processing sample '060_04PI1.rem'...

> Error: Failed to find any matching paired-end reads in

> './samples_all_PE/060_04PI1.rem.2.fq'.
> Aborted.

> Error: Failed to find any matching paired-end reads in

> './samples_all_PE/060_03PI1.rem.2.fq'.
> Aborted.

> Error: Failed to find any matching paired-end reads in

> './samples_all_PE/060_01PI1.rem.2.fq'.
> Aborted.

> Error: Failed to find any matching paired-end reads in

> './samples_all_PE/060_02PI1.rem.2.fq'.
> Aborted.

[Yu Feng]

Hi Julian,

I also have this problem in the latest version of Stacks. 

When I ran tsv2bam, the error is as below

Processing sample 'CJZ05'...

Processing sample 'CJZ17'...

Error: Failed to find any matching paired-end reads in '../cleandata/processed/BX05.2.fq.gz'.

Aborted.

Error: Failed to find any matching paired-end reads in '../cleandata/processed/BX04.2.fq.gz'.

I have run process_radtags, and after that, the Illumina sequence name is as below

E00300:65:HMM3MALXX:6:1110:7019:34465 1:N:0/1

E00300:65:HMM3MALXX:6:1110:6918:34817 1:N:0/1

What should I do?

Yu Feng 

[Julian Catchen]

Hi Yu Feng,

I don't entirely understand your message. The tsv2bam program is telling
you that it cannot find the paired-end file for samples BX04 and BX05.
Do you have paried-end data for these samples, are you sure the
paired-end files aren't empty?

julian

Yu Feng wrote on 9/8/18 8:14 PM:

[Norah Saarman]

Hi Feng,

I am having a similar problem when I try to use the process_radtags output as the input for BWA. Here is a command that should remove the /1 and /2:

zcat sample.1.fq.gz | sed 's/\/1$//g' > sample.new.1.fq

gzip sample.new.1.fq

zcat sample.2.fq.gz | sed 's/\/2$//g' > sample.new.2.fq

gzip sample.new.2.fq

However, it looks like there are other differences in the headers between the matched pairs, which is also what I have found to be a problem  in my case and am waiting to hear back about from Julian (see thread "Demultiplexing ddRAD data with process-radtags doesn't correctly assign F and R reads").

I hope this helps,

Norah Saarman

[Yu Feng]

Hi Julian,

I am sure the paired-end reads are not empty. When I remove the /1,/2 in the reads headers as suggested by Norah, then I got error information like this:

tsv2bam v2.2, executed 2018-09-16 09:40:24

/usr/local/bin/tsv2bam -P . -t 8 -M ./popmap -R /home/biosys/Desktop/FY/Dipteronia_radseq/cleandata/processed

Configuration for this run:

  Stacks directory: './'

  Population map: './popmap'

  Num. samples: 110

  Paired-end reads directory: '/home/biosys/Desktop/FY/Dipteronia_radseq/cleandata/processed/'

  Multithreaded.

Paired-end reads files found, e.g. '/home/biosys/Desktop/FY/Dipteronia_radseq/cleandata/processed/542.2.fq.gz'.

Loading the catalog...

Processing sample '542'...

Processing sample 'BGC12'...

Processing sample '551'...

Processing sample 'BGC03'...

Processing sample 'BGC02'...

Processing sample 'BGC04'...

Processing sample 'BGC16'...

Processing sample '544'...

Error: Unrecognized read name format: expected 'E00300:65:HMM3MALXX:5:1101:10003:10029 2:N:0' to end with /1, /2, _1 or _2.

Aborted.

Error: Unrecognized read name format: expected 'E00300:65:HMM3MALXX:5:1101:10003:14353 2:N:0' to end with /1, /2, _1 or _2.

Error: Unrecognized read name format: expected 'E00300:65:HMM3MALXX:5:1101:10003:29402 2:N:0' to end with /1, /2, _1 or _2.

Aborted.

Aborted.

Error: Unrecognized read name format: expected 'E00515:305:HJYCVCCXY:3:1101:10003:37313 2:N:0' to end with /1, /2, _1 or _2.

Aborted.

Error: Unrecognized read name format: expected 'E00515:305:HJYCVCCXY:3:1101:10003:12138 2:N:0' to end with /1, /2, _1 or _2.

Aborted.

Error: Unrecognized read name format: expected 'E00515:305:HJYCVCCXY:3:1101:10003:11294 2:N:0' to end with /1, /2, _1 or _2.

Error: Unrecognized read name format: expected 'E00300:65:HMM3MALXX:6:1101:10003:16287 2:N:0' to end with /1, /2, _1 or _2.

Aborted.

Aborted.

Error: Unrecognized read name format: expected 'E00300:65:HMM3MALXX:6:1101:10003:15127 2:N:0' to end with /1, /2, _1 or _2.

Aborted.

➜

Any suggestions?

Thanks for your reply.

Yu Feng

在 2018年9月11日星期二 UTC+8上午12:51:01，Julian Catchen写道：

[Yu Feng]

Hi Julian,

Here is the previous log information when I ran tsv2bam:

➜  stacks18.9.5 tsv2bam -P ./ -M ./popmaps/popmap -R ../cleandata/processed/ -t 8

Logging to './tsv2bam.log'.

Configuration for this run:

  Stacks directory: './'

  Population map: './popmaps/popmap'

  Num. samples: 110

  Paired-end reads directory: '../cleandata/processed/'

  Multithreaded.

Paired-end reads files found, e.g. '../cleandata/processed/542.2.fq.gz'.

Loading the catalog...

Processing sample '544'...

Processing sample 'BGC04'...

Processing sample 'BGC12'...

Processing sample 'BGC03'...

Processing sample 'BGC16'...

Error: Failed to find any matching paired-end reads in '../cleandata/processed/551.2.fq.gz'.

Aborted.

Error: Failed to find any matching paired-end reads in '../cleandata/processed/542.2.fq.gz'.

Aborted.

Error: Failed to find any matching paired-end reads in '../cleandata/processed/544.2.fq.gz'.

Aborted.

Error: Failed to find any matching paired-end reads in '../cleandata/processed/BGC04.2.fq.gz'.

Aborted.

Error: Failed to find any matching paired-end reads in '../cleandata/processed/BGC02.2.fq.gz'.

Error: Failed to find any matching paired-end reads in '../cleandata/processed/BGC12.2.fq.gz'.

Aborted.

Aborted.

Error: Failed to find any matching paired-end reads in '../cleandata/processed/BGC16.2.fq.gz'.

Error: Failed to find any matching paired-end reads in '../cleandata/processed/BGC03.2.fq.gz'.

Aborted.

Aborted.

➜  stacks18.9.5 zcat ../cleandata/processed/542.2.fq.gz | head

@E00300:65:HMM3MALXX:5:1101:10003:29402 2:N:0/2

ACGAGTCATCATTAGTGATGGAGGGAGTCATTTCTGTAACAAGGTTTTAGGCAACCTATTGAAGAAGTATGGGGTCCGTCACAAGGTTGCTACACCATATCACCCATAAACATCGGGACAGGTTGAAGTGAGTAATAGGCACATCAAGCA

+

AAFFFJJJJJFAAJJJJJFJJJJJJJAJAAJJJJJJJJJJJJJJJJJJJJFJJFFJAJFFJJJJFAFJJJJFJJJJJJJJFJJJJJJFJJJJJJJJJJJJFJJJJJFJJJJJJJJJFAFJJFJJAAJJJF<J<-<FFJFAJJAFAJFJAJ

@E00300:65:HMM3MALXX:5:1101:10003:43290 2:N:0/2

TGGTGAAATAGCTTACCAACAATGTTTACGGAAGCAAAGGCCTGCATCAAAACGGGATAAATATGACTTTCATTGGATATGAGCTTTTACATCGAGATATGTGGAATTCAAAACTCGCTTTTTGAATTAGACCCAAATGGACCTGGTCAG

+

AAFFFFJJJJJJJJJJJJJFJJJJAFJJJJJJFJJJJJJJJAJJJJJJJJJJJJJJJJJJJJJJJFJFJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJFJJFJJJJJJJJJJJJJJJJJJFJAFJJJJFFJJJJFFJJJ

@E00300:65:HMM3MALXX:5:1101:10003:44064 2:N:0/2

CTAGATAAATACAAGGAATTATTTATCTAATCAAATTAGGAAGTCTTTAAGATAGTCACAAATACACATGAGTTGGATTAGGGTTTGTATTCTTGGAAAGAAAGGAGTCTAAGTGAAGCACAAAATCCTCCCTACACAAGGCAAGGATTT

➜  stacks18.9.5 zcat ../cleandata/processed/542.1.fq.gz | head

@E00300:65:HMM3MALXX:5:1101:10003:29402 1:N:0/1

AATTCTTCCAACTCATTCAACTGATGGCATCTCACTTCACCGGCCTTATCATATTCCCAATTCAACTTCTTGATTGCCCACCAAGCTCTATGTTCTAACTCAACTGGAAGGTGGCATGCCTTTCCAAAAACTAACCTAAACGG

+

JJ<JJJJFJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJFJJJJJ7JJJJFJJJJJJJJJJJJJJJFJJ<JJJJJJJJJJJJJJJJJJJJJJJJJFJJJJJJJJJJ7

@E00300:65:HMM3MALXX:5:1101:10003:43290 1:N:0/1

AATTCCACCATTACCATTATTCATTTCTGAAGTGTTGCAAATCCCATCCATGGAACCATTAAGCTTGTCAACAGAAGATGCTGCGGCGCCTTCTCTTAAGGCTATAGGTGAACTGAGTGAGGCAACACCACCTGTATGAAAAA

+

JJJJAJJJJJJJJJJJJJJJJJJJFJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJFJJJJJJJJFJJJJJJJJFJJJJJJJJJFJJJJJJJJJJJJJ

@E00300:65:HMM3MALXX:5:1101:10003:44064 1:N:0/1

AATTCACAATCAATAAGATTGAAACTCGTGTGAACCTTATTTTGAAAGTGTGCACAAAGCCTTTTGATGTAAAAGCTTATGAGTGCACTATCAAAATAATTCTTTCTCTCCTTACTACTCTATGGCCGAAAATCACACTAGGG

在 2018年9月16日星期日 UTC+8上午10:13:28，Yu Feng写道：

[Felipe Torquato]

Hi Julian,

I am getting the same Error message.

tsv2bam "Error: Failed to find any matching paired-end reads in"

What do I need to do to not to push the remainders through the pipeline?

Do I need to modify anything in the command line of denovo_map.pl?

denovo_map.pl -T 10 -M 2 -n 1 --samples ./process_radtags/ --popmap ./popmap.txt -o ./denovo_map.pl/ --paired

Best,

Felipe

[Rochette de Lempdes, Nicolas Charles]

Hi Felipe,

We need the entire error, if not the entire log, to be able to guess the cause of the error. Otherwise we can't go further than what the program has already told you; it isn't able to match your forward and reverse reads.

Best,

Nicolas

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[Felipe Torquato]

Hi Niolas,

thanks for the quick reply. The Error is the same that people have been described in this topic.

I have paired-end data, and when the denovo_map.pl gets to the stage of tsv2bam I get this error in the log file (see below). I figure out the source of error but I do not know how to solve it.

Julian suggested to not push the remainders through the pipeline. How can I do so?

Thanks in advance!

 

tsv2bam

==========

/groups/hologenomics/software/stacks/v2.2/bin/tsv2bam -P ./denovo_map.pl  -t 10 -M ./popmap.txt -R ./process_radtags/

Logging to './denovo_map.pl/tsv2bam.log'.

Configuration for this run:

  Stacks directory: './denovo_map.pl/'

  Population map: './popmap.txt'

  Num. samples: 94

  Paired-end reads directory: './process_radtags/'

  Multithreaded.

Paired-end reads files found, e.g. './process_radtags/QAA1.2.fq.gz'.

Loading the catalog...

Processing sample 'QAA2'...

Processing sample 'QFD3'...

Processing sample 'QBU1'...

Processing sample 'QFD14'...

Processing sample 'QFD12'...

Processing sample 'QAA1'...

Processing sample 'QAA3'...

Processing sample 'QFD15'...

Processing sample 'QFD2'...

Processing sample 'QFD1'...

Error: Failed to find any matching paired-end reads in './process_radtags/QFD14.2.fq.gz'.

Aborted.

Error: Failed to find any matching paired-end reads in './process_radtags/QFD1.2.fq.gz'.

Aborted.

Error: Failed to find any matching paired-end reads in './process_radtags/QFD3.2.fq.gz'.

Aborted.

Error: Failed to find any matching paired-end reads in './process_radtags/QFD12.2.fq.gz'.

Aborted.

Error: Failed to find any matching paired-end reads in './process_radtags/QBU1.2.fq.gz'.

Aborted.

Error: Failed to find any matching paired-end reads in './process_radtags/QAA3.2.fq.gz'.

Aborted.

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[Felipe Torquato]

Hi Yu,

did you solve it?

Best,

Felipe

[Amy Welsh]

I'm wondering as well if this problem was solved.  I'm also having the problem of tsv2bam failing to find any matching paired-end reads when I get to this point in the denovo_map.pipeline.  I've tried it both within the denovo_map pipeline and running tsv2bam by itself... I get the same error both ways.

Thanks!

Amy Welsh

[Catchen, Julian]

Hi Amy,

In these earlier messages the individuals either did not run their data
through process_radtags, so the fastq headers weren't as expected by
tsv2bam, or they were trying to load samples without any data in the
paired-end file.

Without seeing an error message or your command lines I don't know what
is going on in your case.

julian

Amy Welsh wrote on 2/9/19 4:58 PM:

[Catchen, Julian]

Hi Amy,

If you look at the headers of the files, you can see the problem

Amy Welsh wrote on 2/11/19 3:57 PM:
> the problematic .1 files (CKA6):
>
> @263_5_1101_1637_2214/1/1
> TGCAGCTGGTCACAGAGACCCTCGATGCCACTCTGCATGCCTCCACCACACCTTCAACATCTGCCTGCCCGAATGCCTGGAGGGACACCA
> +
> FFFFFFFFFIIIIIIIIIIFFIIIIIIFFFFIIIIIIIIIIIIIIIIFIIIIIIIIIIIIFFFFFFFFFFFFFFFFFFFFBFFFFFFFFB
>
...
> And here's the corresponding .2 file:
>
> @263_5_1101_1637_2214/2/1
> CGGTGGACCAGGTCATGGGGAGGAGCTTCATAGGCTCAAGAAACTGGACAGATGGGCAGCCAGCCCTCCCAGCTAGACGCTGCTTTACTC
...

The files should have an ID followed by "/1" for the single-ends and the
same ID with "/2" for the paired-end. You can see that yours has an
additional "/1" after the "/2" which is causing the parser to read those
as single-end reads. It seems you potentially ran the files through
process_radtags twice?

Best,

julian

[Felipe Torquato]

Hi Julian,

this is not my case!

Please, se my reads after the process_radtags:

FORWARD:

@170_3_1101_2077_1332/1

TGCAGTCAGAAGAGCGAAAACAACAACTGCGGACATAAAGAACAGCATTCGCCGCAAAACGACCATATCACGTTGACACACGTTTTCAAG

+

JJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJFJJJJJJJFJJJJJJ

@170_3_1101_4330_1332/1

TGCAGTTTAGATTTAGAGCAATATCCTTGTTTTGGTTAATATTGATGGCTGATTAGTAATTCGATTCTTCTCTTATTATATGAGAAGAGA

REVERSE:

@170_4_1101_5477_1314/2

TGTCCTAGGATGCAGACGCCTGGGGGCCCAATAGGAACAGTTTTTGCCAGTGAAAAAGATGATGCTTACACCAGTTTTTCCCACGTTTTC

+

AAFFFJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJFJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJFJJJJJJJJJJJJJJJ

@170_4_1101_7710_1314/2

ATGAAGGCAGTGCAGTAAAACAGTGATTAAGGCTAGTCCAAGAGGAGGACATTACATTTTACAGTATCTTATACCATTCACACATCTCAG

ustacks, cstacks and sstacks work normally. However, when it starts running tsv2bam I always get the same error: 

Paired-end reads files found, e.g. './felipe/sample_01.2.fq.gz'.

Loading the catalog...

Processing sample 'sample_01'...

Processing sample 'sample_03'...

Processing sample 'sample_02'...

Error: Failed to find any matching paired-end reads in './felipe/sample_02.2.fq.gz'.

Aborted.

Error: Failed to find any matching paired-end reads in './felipe/sample_03.2.fq.gz'.

Aborted.

Error: Failed to find any matching paired-end reads in './felipe/sample_01.2.fq.gz'.

Aborted.

Do you have any idea about where my mistake is?

Thanks once more.

Felipe

[Catchen, Julian]

Hi Felipe,

Are your read pairs in phase in the paired files? Probably just a matter
of what you cut/pasted below, but the FASTQ IDs should match in terms of
order between the two files (save the /1 or /2 at the end of the header).

ustacks, cstacks, sstacks only look at the single-end reads, so it is
not a surprise they complete okay, given some problem with the paired-ends.

julian

Felipe Torquato wrote on 2/14/19 4:55 AM:

> Hi Julian,
>
> this is not my case!
> Please, se my reads after the process_radtags:
>

> *FORWARD:*

> @170_3_1101_2077_1332/1
> TGCAGTCAGAAGAGCGAAAACAACAACTGCGGACATAAAGAACAGCATTCGCCGCAAAACGACCATATCACGTTGACACACGTTTTCAAG
> +
> JJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJFJJJJJJJFJJJJJJ
> @170_3_1101_4330_1332/1
> TGCAGTTTAGATTTAGAGCAATATCCTTGTTTTGGTTAATATTGATGGCTGATTAGTAATTCGATTCTTCTCTTATTATATGAGAAGAGA
>
>

> *REVERSE:*

[xu1573...@gmail.com]

Hi Pedro,

I get the same peoblem with you. I kown the key is on the header of the paired reads with 2:N:0 different from 1:N:0. So, can you please give advice on replacing all headers in the paired reads quckily?

Best,

Xu

在 2018年1月25日星期四 UTC+8下午6:15:03，Pedro写道：

[Bartosz Ulaszewski]

Hi Xu,

I have posted solution for the errors when using the --paired parameter here:

https://groups.google.com/forum/#!topic/stacks-users/qvtQhPhXgT4

Best regards,

Bart

[Pedro]

Hi

I'm sorry, I was not actively following the topic...

I understand you already have a solution from Bartosz but I'll share mine also here. I have use a pipe with awk to modify the headers on both -ends files. The headers have a single space separating most info to the small part (1:N:0) that gives the problem. So I have use this to eliminate that part of the header with the pipe:

zcat  FILE.fq.gz | awk  -F " " '{if (NR%4 == 1) print $1; else print $0}' | gzip > NEWFILE.fq.gz

(replace CAPITALS with your own files). I stress that this might not be a universal solution, specially if you have extra spaces on the headers. I did that before process_radtags so it can add the /1 /2 to the header. Also, I placed it on a for loop over all files I had, maintaining the file name but exporting to a different folder.

hope it helps

Pedro