multiple_split_libraries_fastq - Store quality scores?

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Alyse

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Jun 2, 2017, 7:53:58 PM6/2/17
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Hello, 

Is it possible to store quality scores when using multiple_split_libraries_fastq? 

I am using the command: 
multiple_split_libraries_fastq.py -i ./FastqFiles -o ./FilteredFiles -m sampleid_by_file -p parameters.txt

My parameters file is as follows:
 
#quality filtering of joined PE reads
split_libraries_fastq.py:store_qual_scores True #keep .qual file
split_libraries_fastq.py:phred_quality_threshold 19 #minimum quality score of 20
split_libraries_fastq.py:max_bad_run_length 3 #allows 3 poor quality bases before read truncation 
split_libraries_fastq.py:min_per_read_length_fraction 0.74 #minumum consecutive high quality base calls to include in a read as fraction of input length

However, when I run the command it does not output any .qual files (although all the other outputs look fine). Any thoughts on how to fix this issue? I am using MacQIIME 1.9.1-20150604 

Thank you for your assistance!

Alyse

Colin Brislawn

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Jun 2, 2017, 10:33:39 PM6/2/17
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Hello Alyse,

Great question. I think you are on the right track. Try removing the '.py' from your parameter file. Take this one line as an example: 
split_libraries_fastq:store_qual_scores True #keep .qual file

That should pass the parameters to get the results you want, including .fastq files as outputs.

Let me know how well that works for you,
Colin

Alyse

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Jun 5, 2017, 2:56:34 PM6/5/17
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Hi Colin, 

That worked great, thank you! However, I have a follow up question. 

I successfully created the seqs.fna and seqs.qual files with my command (multiple_split_libraries_fastq.py -i ./FastqFiles -o ./FilteredFiles -m sampleid_by_file -p parameters.txt). For my next step I wanted to recombine them into a .fastq file. However, when I use the following command I get an error message. 

convert_fastaqual_fastq.py -f seqs.fna -q seqs.qual -c fastaqual_to_fastq


Traceback (most recent call last):

  File "/macqiime/anaconda/bin/convert_fastaqual_fastq.py", line 113, in <module>

    main()

  File "/macqiime/anaconda/bin/convert_fastaqual_fastq.py", line 110, in main

    full_fasta_headers)

  File "/macqiime/anaconda/lib/python2.7/site-packages/qiime/convert_fastaqual_fastq.py", line 43, in convert_fastaqual_fastq

    full_fastq, full_fasta_headers)

  File "/macqiime/anaconda/lib/python2.7/site-packages/qiime/convert_fastaqual_fastq.py", line 100, in convert_fastq

    MinimalQualParser(qual_file)):

  File "/macqiime/anaconda/lib/python2.7/site-packages/qiime/parse.py", line 759, in MinimalQualParser

    "Invalid qual file. Check the format of the qual files.")

qiime.parse.QiimeParseError: Invalid qual file. Check the format of the qual files.


Any clues as to why I can't recombine the .fna and .qual files? It seems like the .qual output from multiple_split_libraries_fastq.py is not compatible with the convert_fastaqual_fastq.py. 


Thanks again! 


Alyse

Colin Brislawn

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Jun 5, 2017, 3:42:19 PM6/5/17
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Hello Alyse,

Ah, I think I know how to do this elegantly.
 
I wanted to recombine them into a .fastq file. 
OK, try using store_demultiplexed_fastq instead of store_qual_scores that way your outputs are already in the fastq format, and you don't have to deal with .qual files at all.

The fasta / qual file format is old, and used by the 454 sequencer. Just using fastq files should make this easier.

Once you get all these fastq files, you can copy them into a single file using the linux 'cat' command. 

Let me know how well this works for you,
Colin


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