"Could not find any reads in example_sample1-ClassII-2nditeration_2.fq"

[NicoleThue]

Hello, I'm trying to get the pipeline up and running with the example dataset, but I keep getting this error that are no alignments in the bowtie log. Because of this error it doesn't produce the ClassII-2nditeration_2.fq files and keeps the pipeline from completing. This happens right in the beginning, so it likely has to do with the seq2_HLA scripts, but I'm just not sure where the issue is coming from.

[ShirAmit96]

I'm having the same issue Nicole mentioned. If anyone has a solution, please let me know.

[RENXI-NUS]

Sorry for the late reply. I rerun the seq2_HLA script and cannot reproduce your error. Did you use python 2 to call the seq2HLA scripts? Also you need to install biopython for running seq2HLA script using something like "conda install biopython". If still cannot work, can you try your own paired end RNAseq dataset to see if you still observe the error. Thanks.

[jeremymchacon]

Hi @RENXI-NUS, cool pipeline! I also get the same warning as @NicoleThue, although I assumed that the example data simply didn't have any class 2 HLA reads. This is what the top part of my log looks like:

`rename: not enough arguments

Usage: rename [options] expression replacement file...

Options: -v, --verbose explain what is being done -s, --symlink act on symlink target

-h, --help display this help and exit -V, --version output version information and exit

For more details see rename(1).

reads processed: 1677

reads with at least one reported alignment: 1677 (100.00%)

reads that failed to align: 0 (0.00%)

Reported 119961 paired-end alignments to 1 output stream(s) Warning: Could not find any reads in "/home/riss/chaco001/pasram/test_output/example/example_sample1/example_sample1-ClassII-2nditeration_1.fq" Warning: Could not find any reads in "/home/riss/chaco001/pasram/test_output/example/example_sample1/example_sample1-ClassII-2nditeration_2.fq"

reads processed: 0

reads with at least one reported alignment: 0 (0.00%)

reads that failed to align: 0 (0.00%)

No alignments Now running seq2HLA version 2.2.! Input is a gipped file ..... The read length of your input fastq was determined to be 48, so 1 mismatches will be allowed and 12 threads will be used by bowtie. ----------HLA class I------------ First iteration starts.... Mapping ......

reads processed: 114736

reads with at least one reported alignment: 5131 (4.47%)

reads that failed to align: 109605 (95.53%)

Reported 420338 paired-end alignments to 1 output stream(s)

Calculation of first digital haplotype..... The digital haplotype is written into /home/riss/chaco001/pasram/test_output/example/example_sample1/example_sample1-ClassI.digitalhaplotype1 1st iteration done. Now removing reads that mapped to the three top-scoring groups ....... Second iterations starts ..... Mapping ...... Calculation of second digital haplotype..... The digital haplotype is written into /home/riss/chaco001/pasram/test_output/example/example_sample1/example_sample1-ClassI.digitalhaplotype2 2nd iteration done. -----------2 digit typing results-------------

Locus Allele 1 Confidence Allele 2 Confidence

A A02 1.914287e-05 A11 0.001489945 B B67 0.1258518 B15 0.004450812 C C07 0.01216089 C08 0.02312405 Calculation of locus-specific expression ... /home/riss/chaco001/pasram/test_output/example/example_sample1/example_sample1-ClassI.bowtielog A: 15867.78 RPKM C: 25236.48 RPKM B: 2273.1 RPKM -----------4 digit typing results-------------

Locus Allele 1 Confidence Allele 2 Confidence

!A A02:03' 1.914287e-05 A11:02' 0.001489945 !B B67:02 0.1258518 B15:25' 0.004450812 !C C07:02 0.01299892 C08:01 0.02312405 ----------HLA class II------------ ClassII: first iteration starts.... Mapping ......

reads processed: 114736

reads with at least one reported alignment: 0 (0.00%)

reads that failed to align: 114736 (100.00%)

No alignments

ClassII: calculation of first digital haplotype..... The digital haplotype is written into /home/riss/chaco001/pasram/test_output/example/example_sample1/example_sample1-ClassII.digitalhaplotype1 1st iteration done. Now removing reads that mapped to the three top-scoring groups ....... Nothing to remove `

Note that it finds and aligns class I HLA reads, but not class II. Note also the failed rename at the top, which I assume/hope is not critical?

Would it be possible to include the log file that you get when you run the example data? That would help me troubleshoot some other issues I have been having. Thanks!

[RENXI-NUS]

Hello! Sure. I attach the log file here for your reference. log.txt