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Old 02-16-2014, 11:00 AM   #1
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Location: USA

Join Date: Feb 2014
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Default bowtie2 Saw ASCII character 7 but expected 33-based Phred qua

I'm running bowtie2 on a windows 7, 64 bit machine. My reads are prokaryotic paired end reads derived from an Illumina HiSeq instrument

below are the outputs for 2 alignments:


C:\bowtie2>perl bowtie2 --very-fast-local -t -p2 -x genomeindex -1 R:\OperonNGSdata\run_data\A-1_GGACCC_L008_R1_001.fastq -2 R:\OperonNGSdata\run_data\A-1_GGACCC_L008_R2_001.fastq -S A1_Alignment.sam
Time loading reference: 00:00:00
Time loading forward index: 00:00:00
Time loading mirror index: 00:00:00
Multiseed full-index search: 02:06:00
24462238 reads; of these:
24462238 (100.00%) were paired; of these:
1566890 (6.41%) aligned concordantly 0 times
5594011 (22.87%) aligned concordantly exactly 1 time
17301337 (70.73%) aligned concordantly >1 times
1566890 pairs aligned concordantly 0 times; of these:
156916 (10.01%) aligned discordantly 1 time
1409974 pairs aligned 0 times concordantly or discordantly; of these:
2819948 mates make up the pairs; of these:
2064379 (73.21%) aligned 0 times
45713 (1.62%) aligned exactly 1 time
709856 (25.17%) aligned >1 times
95.78% overall alignment rate
Time searching: 02:06:00
Overall time: 02:06:00

Result2 :

C:\bowtie2>perl bowtie2 --very-fast-local -t -p2 -x genomeindex -1 R:\OperonNGSdata\run_data\A-2_TTCAGC_L008_R1_001.fastq -2 R:\OperonNGSdata\run_data\A-2_TTCAG
C_L008_R2_001.fastq -S A2_Alignment.sam
Time loading reference: 00:00:00
Time loading forward index: 00:00:01
Time loading mirror index: 00:00:00
Saw ASCII character 7 but expected 33-based Phred qual.
terminate called after throwing an instance of 'int'

This application has requested the Runtime to terminate it in an unusual way.
Please contact the application's support team for more information.
bowtie2-align exited with value 255

As can be seen, the first run completes successfully, while the second one quits unexpectedly after throwing an error. This is confusing since the samples are replicates processed in exactly the same way on the same instrument and (presumably) identically processed according to the latest version of the Illumina pipeline (version ??).

Last edited by swatve3; 02-16-2014 at 11:35 AM.
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Old 02-16-2014, 01:54 PM   #2
Peter (Biopython etc)
Location: Dundee, Scotland, UK

Join Date: Jul 2009
Posts: 1,543

It could be the second FASTQ file is corrupt (there shouldn't be an ASCII char 7 in it). I would start by checking the contents of the FASTQ looking for this bad character (can you program? This would be easy in Perl/Python/Ruby etc).

You could also check the MD5 checksum (if available), or try re-downloading the FASTQ file in case it was a network glitch or hard drive error to blame.
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Old 02-16-2014, 02:53 PM   #3
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Thanks for replying.

Some more info:

1) I've mapped these files to the genome using CLC genomics workbench before without any glitch
2) I cannot program in Perl/Python/Ruby without going through a lot of pain and anguish
3) I'll try to download the file again and try a second run to see if that works
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Old 02-16-2014, 05:24 PM   #4
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Location: East Coast USA

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Posts: 7,077

Perhaps CLC is more tolerant of a malformed fastq record and skips it.

You can use one of these scripts to see if they can find the bad fastq record.
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bowtie2 output, error, phred quality

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