split_libraries_fastq.py error :Nonn-header line passed as input. Header must start '@'

[Shi Ming Tan]

Hello Qiime experts,

I was trying to split some fastq files using the split_libraries_fastq.py and I have come up with an error:

qiime@qiime-190-virtual-box:~/Desktop/qiime/mi-seq/000000000-APU1H_895zop9hL1/trimmed_duk/merge-sortmerna/sortmerna/rRNA$ split_libraries_fastq.py -i ACC291215100A_R1R2_merge_rRNA.fastq,ACC291215100B_R1R2_merge_rRNA.fastq,ACC291215100C_R1R2_merge_rRNA.fastq,ACC29121510A_R1R2_merge_rRNA.fastq,ACC29121510B_R1R2_merge_rRNA.fastq,ACC29121510C_R1R2_merge_rRNA.fastq,ACC2912151KA_R1R2_merge_rRNA.fastq,ACC2912151KB_R1R2_merge_rRNA.fastq,ACC2912151KC_R1R2_merge_rRNA.fastq,ACC291215SF_R1R2_merge_rRNA.fastq,ACC291215SP_R1R2_merge_rRNA.fastq,B4R1301215A_R1R2_merge_rRNA.fastq,B4R1301215B_R1R2_merge_rRNA.fastq,B4R1301215C_R1R2_merge_rRNA.fastq,B4R86190216A_R1R2_merge_rRNA.fastq,B4R86190216B_R1R2_merge_rRNA.fastq,B4R86290116A_R1R2_merge_rRNA.fastq,B4R86290116B_R1R2_merge_rRNA.fastq,B4R86290116C_R1R2_merge_rRNA.fastq,B4UPWRP090316A_R1R2_merge_rRNA.fastq,B4UPWRP090316B_R1R2_merge_rRNA.fastq,B4UPWRP090316C_R1R2_merge_rRNA.fastq,B4UPWRP210116A_R1R2_merge_rRNA.fastq,B4UPWRP210116B_R1R2_merge_rRNA.fastq,B4UPWRP210116C_R1R2_merge_rRNA.fastq,B4UPWRP230316A_R1R2_merge_rRNA.fastq,B4UPWRP230316B_R1R2_merge_rRNA.fastq,B4UPWRP230316C_R1R2_merge_rRNA.fastq,R86190216100A_R1R2_merge_rRNA.fastq,R86190216100B_R1R2_merge_rRNA.fastq,R8619021610A_R1R2_merge_rRNA.fastq,R8619021610B_R1R2_merge_rRNA.fastq,R8619021610C_R1R2_merge_rRNA.fastq,R861902161A_R1R2_merge_rRNA.fastq -o split_library -m mapping.txt --sample_ids ACC291215100A,ACC291215100B,ACC291215100C,ACC29121510A,ACC29121510B,ACC29121510C,ACC2912151KA,ACC2912151KB,ACC2912151KC,ACC291215SF,ACC291215SP,B4R1301215A,B4R1301215B,B4R1301215C,B4R86190216A,B4R86190216B,B4R86290116A,B4R86290116B,B4R86290116C,B4UPWRP090316A,B4UPWRP090316B,B4UPWRP090316C,B4UPWRP210116A,B4UPWRP210116B,B4UPWRP210116C,B4UPWRP230316A,B4UPWRP230316B,B4UPWRP230316C,R86190216100A,R86190216100B,R8619021610A,R8619021610B,R8619021610C,R861902161A -r 3 -p 0.75 -n 0 -q 19 --barcode_type 'not-barcoded'

Traceback (most recent call last):

  File "/usr/local/bin/split_libraries_fastq.py", line 365, in <module>

    main()

  File "/usr/local/bin/split_libraries_fastq.py", line 344, in main

    for fasta_header, sequence, quality, seq_id in seq_generator:

  File "/usr/local/lib/python2.7/dist-packages/qiime/split_libraries_fastq.py", line 239, in process_fastq_single_end_read_file_no_barcode

    phred_offset=phred_offset):

  File "/usr/local/lib/python2.7/dist-packages/qiime/split_libraries_fastq.py", line 278, in process_fastq_single_end_read_file

    post_casava_v180 = is_casava_v180_or_later(fastq_read_f_line1)

  File "/usr/local/lib/python2.7/dist-packages/qiime/parse.py", line 37, in is_casava_v180_or_later

    "Non-header line passed as input. Header must start with '@'."

AssertionError: Non-header line passed as input. Header must start with '@'.

The files have already been adapter- and quality- trimmed. 

I have tried with it with the first 5 files and it seemed to work.

Could anyone please advice?

Thank You!

[Barvaz Sini]

Hi,

it seems from the error message one of the fastq files has a sequence header that does not start with a '@' symbol. Are you sure all the files you are using for input are fastq files?

One option would be to try to split the list of files in half and try to run the command on both, and to see which one (or both) gives you the error, and continue this until you find the problematic files(s)?

Can you also paste the list of commands you applied to your files (starting from the raw read fastq files, up to using them as input to split_libraries)?

Thanks

Amnon

[Mark Strong]

Hi Shi,

Can you check all your merged fastq files have some data in it ? I guess some of your fastq files might be just empty.  

Cheers,

[Shi Ming Tan]

Dear Amnon and Mark,

Thanks for the replies! I have managed to solve the problem. :)

It was due to a file that was empty!

Regards

Shi Ming