Hello Tim,

Yes, this is a known issue, a few of the tools do not function with sequences with the newer formatted identifiers.


There is a bare-bones Trello ticket for the upgrade, but to be honest, this hasn't been prioritized because of so few use cases:
https://trello.com/c/bhurghHk

After running "FASTQ Groomer", doing some QC is a good idea by running "FastQC", then " FASTQ Trimmer" or "FASTQ Quality Trimmer" are options. From there, just proceed with mapping. You can always filter the mapping results to restrict hits to those resulting from property paired data after (See "NGS: SAM Tools -> Filter SAM") or certain other tools will have this as an option on the downstream tool form itself.

So, you don't need to filter twice with respect to pairs - once at the end is generally considered OK as long as the input data all meet some minimum quality thresholds (to optimally map with the given tool/parameters and avoid processing problems).

Hopefully this helps,

Jen
Galaxy team

On 4/23/13 8:01 PM, Timothy Brennan wrote:

This question is w/ regards to pre-processing whole genome resequencing data for mapping data to a reference yeast strain.

I'm having trouble joining paired end data. I have two files per sample (read1 and read2).

I've successfully uploaded my fastq.gz files into galaxy using FTP. I have two fastq files for each direction per strain labelled for example:

(for the left hand dir) 130104_7001240_0133_AH0854ADXX.lane_2.CCGTCC.1.fastq

(for the right hand dir) 130104_7001240_0133_AH0854ADXX.lane_2.CCGTCC.2.fastq

Now once I groom each using FASTQ Groomer I'm trying to join them to get a single file and I'm joining 0% of the reads. So I think the header or directory is not in the correct format. E.g., the raw groomed reads for the left hand and right hand look like:

(for the left hand dir) 130104_7001240_0133_AH0854ADXX.lane_2.CCGTCC.1.fastq

@HWI-ST1240:133:H0854ADXX:2:1101:2716:1998 1:N:0:CCGTCC

NGTATGGAAGACGTAGAGTGGATGAAAATTTTGTGAAAAAAAAAAGCTTATAGGAACAAAAACATCCTTACATCTTCGGGTATTTCTTCTAGGGTTGAAGT

+

!!!%%%%%)))))**(*(!$()(((***(***')(**********)'))%!!%&%$$$$$####$$$$$$"$!!""!!##!!!!$$%$""!!"#$#!!!!!

@HWI-ST1240:133:H0854ADXX:2:1101:5045:1994 1:N:0:CCGTCC

NCCAGACACAGTTAACGCAACCTGACATGCAACAGTTATCGGGTTCTTGTGGTTTTGCAGGCACTTGGACACCTGCTATTTTCTTCGTTCCGCCGCTAAGC

(for the right hand dir) 130104_7001240_0133_AH0854ADXX.lane_2.CCGTCC.2.fastq

@HWI-ST1240:133:H0854ADXX:2:1101:2716:1998 2:N:0:CCGTCC
GCATAGTTACTTTTTGATCACTAACAACGATATATTATCGTTGAACAATTTACTACGCAAAACAGTTCACGTGATGTACGTCAGATAATTCACTGAAGGTA
+
$$$''''')))))++++++(+++++++++++++++++*+*++*++++++++++*++++*+++++*))))))''''&'&'%&%%%%$%%%'&%%%%%$%%$!
@HWI-ST1240:133:H0854ADXX:2:1101:5045:1994 2:N:0:CCGTCC
ATGTATTATAAGCCCGAATCAGATACTCAAATTTGAAAAAAGATATCTTTCTCCTCCGACATGGCCGAACTCATTTACATAAATAGCATAAATTAAACAGA

According to the wiki I think the fastq format should look something like this with /1 and /2 corresponding the each paired file.

@61CC3AAXX100125:7:118:2538:5577/1

GACACCTTTAATGTCTGAAAAGAGACATTCACCATCTATTCTCTTGGAGGGCTACCACCTAAGAGCCTTCATCCCC

+

?>CADFEEEBEDIEHHIDGGGEEEEHFFGIGIIFFIIEFHIIIIHIIFFIIIDEIIGIIIEHFFFIIEHIFA@?==

@61CC3AAXX100125:7:1:17320:13701/1

CTCAGAAGACCCTGAGAACATGTGCCCAAGGTGGTCACAGTGCATCTTAGTTTTGTACATTTTAGGGAGATATGAG

+

?BCAAADBBGGHGIDDDGHFEIFIIIIFGEIFIIFIGIGEFIIGGIIHEFFHHHIHEIFGHHIEFIIEECE?>@89

Any suggestions on how to get the files in the correct format/header to be able to join them?

Last question, what is the tool to trim reads based on quality again?

Thanks very much gentle people

Tim



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