# Problem with paired end demo on new install **URL:** \ **Category:** KneadData\ **Created:** [September 11, 2020, 4:16pm UTC](https://forum.biobakery.org/t/problem-with-paired-end-demo-on-new-install/1004 "2020-09-11T16:16:35Z")\ **Posts on this page:** 16\ **Page:** 1
**Author:** ![lowk](https://yyz1.discourse-cdn.com/flex027/user_avatar/forum.biobakery.org/lowk/32/392_2.png) [@lowk](https://forum.biobakery.org/u/lowk)\ **Post date:** [September 11, 2020, 4:16pm UTC](https://forum.biobakery.org/t/problem-with-paired-end-demo-on-new-install/1004/1 "2020-09-11T16:16:35Z")
I have a strange and jarring error. I have just installed kneaaddata on a new cluster for the first time, and I am seeing some strange behaviour. I installed with pip install kneaddata. This is kneaddata v0.7.10, running on CentOS 6.7 with python 2.7.13. When I run it on any paired data (including the demo data), it runs without error but all of the reads end up in the “unmatched\_1.fastq” file. This is what I see after running the paired end command in the tutorial: > bash$ wc seq1\_kneaddata\* > 0 0 0 seq1\_kneaddata\_demo\_db\_bowtie2\_paired\_contam\_1.fastq > 0 0 0 seq1\_kneaddata\_demo\_db\_bowtie2\_paired\_contam\_2.fastq > 0 0 0 seq1\_kneaddata\_demo\_db\_bowtie2\_unmatched\_1\_contam.fastq > 0 0 0 seq1\_kneaddata\_demo\_db\_bowtie2\_unmatched\_2\_contam.fastq > 138 1354 18029 seq1\_kneaddata.log > 0 0 0 seq1\_kneaddata\_paired\_1.fastq > 0 0 0 seq1\_kneaddata\_paired\_2.fastq > 141364 141364 13333610 seq1\_kneaddata.repeats.removed.1.fastq > 141364 141364 13302786 seq1\_kneaddata.repeats.removed.2.fastq > 21540 21540 1899203 seq1\_kneaddata.repeats.removed.unmatched.1.fastq > 3384 3384 289698 seq1\_kneaddata.repeats.removed.unmatched.2.fastq > 141364 141364 13333610 seq1\_kneaddata.trimmed.1.fastq > 141364 141364 13302786 seq1\_kneaddata.trimmed.2.fastq > 21540 21540 1899203 seq1\_kneaddata.trimmed.single.1.fastq > 3388 3388 290085 seq1\_kneaddata.trimmed.single.2.fastq > 141364 141364 13333610 seq1\_kneaddata\_unmatched\_1.fastq > 0 0 0 seq1\_kneaddata\_unmatched\_2.fastq and looking in the log I see > 09/11/2020 04:53:28 PM - kneaddata.utilities - DEBUG: 35341 reads; of these: > 35341 (100.00%) were unpaired; of these: > 35341 (100.00%) aligned 0 times > 0 (0.00%) aligned exactly 1 time I have tried playing around with the sequence identifier lines in the fastq, but it doesn’t seem to have any impact. Am I missing some dependency, or is there some known issue with parsing the fastq files on some systems? ---
**Author:** ![lauren.j.mciver](https://avatars.discourse-cdn.com/v4/letter/l/f05b48/32.png) [@lauren.j.mciver](https://forum.biobakery.org/u/lauren.j.mciver)\ **Post date:** [September 11, 2020, 7:23pm UTC](https://forum.biobakery.org/t/problem-with-paired-end-demo-on-new-install/1004/2 "2020-09-11T19:23:40Z")
Hello, Thank you for the detailed post. I tried out the tutorial files with v0.7.10 and I see reads in the paired output files as expected. The tool and dependencies should operate the same on different operating systems; We run/test on a variety of platforms including Centos (which you are using). Is it possible the read identifiers in the files are of an unexpected format? Just to double check can you re-download the files and try running again. If you see the same issue would you post your log file? Then I can dig in a bit more to try to figure out what might be up. Thank you, Lauren ---
**Author:** ![lowk](https://yyz1.discourse-cdn.com/flex027/user_avatar/forum.biobakery.org/lowk/32/392_2.png) [@lowk](https://forum.biobakery.org/u/lowk)\ **Post date:** [September 18, 2020, 9:15am UTC](https://forum.biobakery.org/t/problem-with-paired-end-demo-on-new-install/1004/3 "2020-09-18T09:15:04Z")
I uninstalled and reinstalled kneaddataa, and redownloaded the data, but still see the same issue. Commands run and log file are below. > bash$ pip install kneaddata > DEPRECATION: Python 2.7 reached the end of its life on January 1st, 2020. Please upgrade your Python as Python 2.7 is no longer maintained. pip 21.0 will drop support for Python 2.7 in January 2021. More details about Python 2 support in pip can be found at [Release process - pip documentation v24.0.dev0](https://pip.pypa.io/en/latest/development/release-process/#python-2-support) > Processing /gfs/home/ljostins/.cache/pip/wheels/92/95/06/8892c06b81ccd85a710524ad1edf5f77b24a5be0faeb89d5b6/kneaddata-0.7.10-cp27-none-any.whl > Installing collected packages: kneaddata > Successfully installed kneaddata-0.7.10 > WARNING: You are using pip version 20.2.1; however, version 20.2.3 is available. > You should consider upgrading via the ‘/gfs/devel/ljostins/python-venv-2.7.13/bin/python -m pip install --upgrade pip’ command. > bash$ wget [https://github.com/biobakery/kneaddata/files/4703820/input.zip](https://github.com/biobakery/kneaddata/files/4703820/input.zip) > –2020-09-18 10:03:46-- [https://github.com/biobakery/kneaddata/files/4703820/input.zip](https://github.com/biobakery/kneaddata/files/4703820/input.zip) > Resolving [github.com](http://github.com)… 140.82.121.4 > Connecting to github.com|140.82.121.4|:443… connected. > HTTP request sent, awaiting response… 302 Found > Location: [https://github-production-repository-file-5c1aeb.s3.amazonaws.com/253871273/4703820?X-Amz-Algorithm=AWS4-HMAC-SHA256&X-Amz-Credential=AKIAIWNJYAX4CSVEH53A%2F20200918%2Fus-east-1%2Fs3%2Faws4\_request&X-Amz-Date=20200918T090347Z&X-Amz-Expires=300&X-Amz-Signature=d029decd647f7ed7e1830c38a3852aac07654bb62ddff9ee60831f7849dc3ee2&X-Amz-SignedHeaders=host&actor\_id=0&key\_id=0&repo\_id=253871273&response-content-disposition=attachment%3Bfilename%3Dinput.zip&response-content-type=application%2Fzip](https://github-production-repository-file-5c1aeb.s3.amazonaws.com/253871273/4703820?X-Amz-Algorithm=AWS4-HMAC-SHA256&X-Amz-Credential=AKIAIWNJYAX4CSVEH53A%2F20200918%2Fus-east-1%2Fs3%2Faws4_request&X-Amz-Date=20200918T090347Z&X-Amz-Expires=300&X-Amz-Signature=d029decd647f7ed7e1830c38a3852aac07654bb62ddff9ee60831f7849dc3ee2&X-Amz-SignedHeaders=host&actor_id=0&key_id=0&repo_id=253871273&response-content-disposition=attachment%3Bfilename%3Dinput.zip&response-content-type=application%2Fzip) [following] > –2020-09-18 10:03:47-- [https://github-production-repository-file-5c1aeb.s3.amazonaws.com/253871273/4703820?X-Amz-Algorithm=AWS4-HMAC-SHA256&X-Amz-Credential=AKIAIWNJYAX4CSVEH53A%2F20200918%2Fus-east-1%2Fs3%2Faws4\_request&X-Amz-Date=20200918T090347Z&X-Amz-Expires=300&X-Amz-Signature=d029decd647f7ed7e1830c38a3852aac07654bb62ddff9ee60831f7849dc3ee2&X-Amz-SignedHeaders=host&actor\_id=0&key\_id=0&repo\_id=253871273&response-content-disposition=attachment%3Bfilename%3Dinput.zip&response-content-type=application%2Fzip](https://github-production-repository-file-5c1aeb.s3.amazonaws.com/253871273/4703820?X-Amz-Algorithm=AWS4-HMAC-SHA256&X-Amz-Credential=AKIAIWNJYAX4CSVEH53A%2F20200918%2Fus-east-1%2Fs3%2Faws4_request&X-Amz-Date=20200918T090347Z&X-Amz-Expires=300&X-Amz-Signature=d029decd647f7ed7e1830c38a3852aac07654bb62ddff9ee60831f7849dc3ee2&X-Amz-SignedHeaders=host&actor_id=0&key_id=0&repo_id=253871273&response-content-disposition=attachment%3Bfilename%3Dinput.zip&response-content-type=application%2Fzip) > Resolving [github-production-repository-file-5c1aeb.s3.amazonaws.com](http://github-production-repository-file-5c1aeb.s3.amazonaws.com)… 52.217.88.100 > Connecting to github-production-repository-file-5c1aeb.s3.amazonaws.com|52.217.88.100|:443… connected. > HTTP request sent, awaiting response… 200 OK > Length: 9856071 (9.4M) [application/zip] > Saving to: `input.zip’ > > 100%[==========================================================================================================================================\>] 9,856,071 3.74M/s in 2.5s > > 2020-09-18 10:03:50 (3.74 MB/s) - `input.zip’ saved [9856071/9856071] > > bash$ unzip input.zip > Archive: input.zip > creating: input/ > inflating: input/.DS\_Store > creating: \_\_MACOSX/ > creating: \_\_MACOSX/input/ > inflating: \_\_MACOSX/input/.\_.DS\_Store > inflating: input/seq2.fastq > inflating: \_\_MACOSX/input/.\_seq2.fastq > inflating: input/seq1.fastq > inflating: \_\_MACOSX/input/.\_seq1.fastq > inflating: input/demo\_db.3.bt2 > inflating: \_\_MACOSX/input/.\_demo\_db.3.bt2 > inflating: input/demo\_db.2.bt2 > inflating: \_\_MACOSX/input/.\_demo\_db.2.bt2 > inflating: input/singleEnd.fastq > inflating: \_\_MACOSX/input/.\_singleEnd.fastq > inflating: input/demo\_db.1.bt2 > inflating: \_\_MACOSX/input/.\_demo\_db.1.bt2 > inflating: input/SE\_extra.fastq > inflating: \_\_MACOSX/input/.\_SE\_extra.fastq > inflating: input/demo\_db.4.bt2 > inflating: \_\_MACOSX/input/.\_demo\_db.4.bt2 > inflating: input/demo\_db.rev.1.bt2 > inflating: \_\_MACOSX/input/.\_demo\_db.rev.1.bt2 > inflating: input/demo\_db.rev.2.bt2 > inflating: \_\_MACOSX/input/.\_demo\_db.rev.2.bt2 > inflating: \_\_MACOSX/.\_input > > bash$ kneaddata --input input/seq1.fastq --input input/seq2.fastq --reference-db input/demo\_db --output kneaddataOutputPairedEnd --trf …/…/software/trf/ > Initial number of reads ( /gfs/archive/jostins/microbiome/kneadata/demo/input/seq1.fastq ): 42473 > Initial number of reads ( /gfs/archive/jostins/microbiome/kneadata/demo/input/seq2.fastq ): 42473 > Running Trimmomatic … > Total reads after trimming ( /gfs/archive/jostins/microbiome/kneadata/demo/kneaddataOutputPairedEnd/seq1\_kneaddata.trimmed.1.fastq ): 35341 > Total reads after trimming ( /gfs/archive/jostins/microbiome/kneadata/demo/kneaddataOutputPairedEnd/seq1\_kneaddata.trimmed.2.fastq ): 35341 > Total reads after trimming ( /gfs/archive/jostins/microbiome/kneadata/demo/kneaddataOutputPairedEnd/seq1\_kneaddata.trimmed.single.1.fastq ): 5385 > Total reads after trimming ( /gfs/archive/jostins/microbiome/kneadata/demo/kneaddataOutputPairedEnd/seq1\_kneaddata.trimmed.single.2.fastq ): 847 > Running trf … > Running trf … > Running trf … > Running trf … > Decontaminating … > Running bowtie2 … > Total reads after removing those found in reference database ( /gfs/archive/jostins/microbiome/kneadata/demo/kneaddataOutputPairedEnd/seq1\_kneaddata\_demo\_db\_bowtie2\_paired\_clean\_1.fastq ): 0 > Total reads after removing those found in reference database ( /gfs/archive/jostins/microbiome/kneadata/demo/kneaddataOutputPairedEnd/seq1\_kneaddata\_demo\_db\_bowtie2\_paired\_clean\_2.fastq ): 0 > Total reads after merging results from multiple databases ( /gfs/archive/jostins/microbiome/kneadata/demo/kneaddataOutputPairedEnd/seq1\_kneaddata\_paired\_1.fastq ): 0 > Total reads after merging results from multiple databases ( /gfs/archive/jostins/microbiome/kneadata/demo/kneaddataOutputPairedEnd/seq1\_kneaddata\_paired\_2.fastq ): 0 > Total reads after removing those found in reference database ( /gfs/archive/jostins/microbiome/kneadata/demo/kneaddataOutputPairedEnd/seq1\_kneaddata\_demo\_db\_bowtie2\_unmatched\_1\_clean.fastq ): 35341 > Total reads after merging results from multiple databases ( /gfs/archive/jostins/microbiome/kneadata/demo/kneaddataOutputPairedEnd/seq1\_kneaddata\_unmatched\_1.fastq ): 35341 > Total reads after removing those found in reference database ( /gfs/archive/jostins/microbiome/kneadata/demo/kneaddataOutputPairedEnd/seq1\_kneaddata\_demo\_db\_bowtie2\_unmatched\_2\_clean.fastq ): 0 > Total reads after merging results from multiple databases ( /gfs/archive/jostins/microbiome/kneadata/demo/kneaddataOutputPairedEnd/seq1\_kneaddata\_unmatched\_2.fastq ): 0 > > Final output files created: > /gfs/archive/jostins/microbiome/kneadata/demo/kneaddataOutputPairedEnd/seq1\_kneaddata\_paired\_1.fastq > /gfs/archive/jostins/microbiome/kneadata/demo/kneaddataOutputPairedEnd/seq1\_kneaddata\_paired\_2.fastq > /gfs/archive/jostins/microbiome/kneadata/demo/kneaddataOutputPairedEnd/seq1\_kneaddata\_unmatched\_1.fastq > /gfs/archive/jostins/microbiome/kneadata/demo/kneaddataOutputPairedEnd/seq1\_kneaddata\_unmatched\_2.fastq > bash$ wc kneaddataOutputPairedEnd/\* > 0 0 0 kneaddataOutputPairedEnd/seq1\_kneaddata\_demo\_db\_bowtie2\_paired\_contam\_1.fastq > 0 0 0 kneaddataOutputPairedEnd/seq1\_kneaddata\_demo\_db\_bowtie2\_paired\_contam\_2.fastq > 0 0 0 kneaddataOutputPairedEnd/seq1\_kneaddata\_demo\_db\_bowtie2\_unmatched\_1\_contam.fastq > 0 0 0 kneaddataOutputPairedEnd/seq1\_kneaddata\_demo\_db\_bowtie2\_unmatched\_2\_contam.fastq > 138 1354 17765 kneaddataOutputPairedEnd/seq1\_kneaddata.log > 0 0 0 kneaddataOutputPairedEnd/seq1\_kneaddata\_paired\_1.fastq > 0 0 0 kneaddataOutputPairedEnd/seq1\_kneaddata\_paired\_2.fastq > 141364 141364 13333610 kneaddataOutputPairedEnd/seq1\_kneaddata.repeats.removed.1.fastq > 141364 141364 13302786 kneaddataOutputPairedEnd/seq1\_kneaddata.repeats.removed.2.fastq > 21540 21540 1899203 kneaddataOutputPairedEnd/seq1\_kneaddata.repeats.removed.unmatched.1.fastq > 3384 3384 289698 kneaddataOutputPairedEnd/seq1\_kneaddata.repeats.removed.unmatched.2.fastq > 141364 141364 13333610 kneaddataOutputPairedEnd/seq1\_kneaddata.trimmed.1.fastq > 141364 141364 13302786 kneaddataOutputPairedEnd/seq1\_kneaddata.trimmed.2.fastq > 21540 21540 1899203 kneaddataOutputPairedEnd/seq1\_kneaddata.trimmed.single.1.fastq > 3388 3388 290085 kneaddataOutputPairedEnd/seq1\_kneaddata.trimmed.single.2.fastq > 141364 141364 13333610 kneaddataOutputPairedEnd/seq1\_kneaddata\_unmatched\_1.fastq > 0 0 0 kneaddataOutputPairedEnd/seq1\_kneaddata\_unmatched\_2.fastq > 756810 758026 71002356 total Here is the logfile: > bash$ cat kneaddataOutputPairedEnd/seq1\_kneaddata.log > > 09/18/2020 10:08:37 AM - kneaddata.knead\_data - INFO: Running kneaddata v0.7.10 > > 09/18/2020 10:08:37 AM - kneaddata.knead\_data - INFO: Output files will be written to: /gfs/archive/jostins/microbiome/kneadata/demo/kneaddataOutputPairedEnd > > 09/18/2020 10:08:37 AM - kneaddata.knead\_data - DEBUG: Running with the following arguments: > > verbose = False > > bypass\_trf = False > > bmtagger\_path = None > > minscore = 50 > > bowtie2\_path = /gfs/apps/bio/bowtie2-2.3.0/bowtie2 > > maxperiod = 500 > > no\_discordant = False > > serial = False > > fastqc\_start = False > > bmtagger = False > > cat\_final\_output = False > > log\_level = DEBUG > > log = /gfs/archive/jostins/microbiome/kneadata/demo/kneaddataOutputPairedEnd/seq1\_kneaddata.log > > sequencer\_source = NexteraPE > > max\_memory = 500m > > remove\_intermediate\_output = False > > fastqc\_path = None > > output\_dir = /gfs/archive/jostins/microbiome/kneadata/demo/kneaddataOutputPairedEnd > > trf\_path = /gfs/archive/jostins/microbiome/software/trf/trf > > remove\_temp\_output = True > > reference\_db = /gfs/archive/jostins/microbiome/kneadata/demo/input/demo\_db > > input = /gfs/archive/jostins/microbiome/kneadata/demo/input/seq1.fastq /gfs/archive/jostins/microbiome/kneadata/demo/input/seq2.fastq > > pi = 10 > > reorder = False > > pm = 80 > > trimmomatic\_path = /gfs/apps/bio/trimmomatic-0.35/trimmomatic-0.35.jar > > store\_temp\_output = False > > mismatch = 7 > > threads = 1 > > delta = 7 > > bowtie2\_options = --very-sensitive --phred33 > > bypass\_trim = False > > processes = 1 > > trimmomatic\_quality\_scores = -phred33 > > fastqc\_end = False > > trimmomatic\_options = None > > output\_prefix = seq1\_kneaddata > > match = 2 > > 09/18/2020 10:08:37 AM - kneaddata.utilities - INFO: READ COUNT: raw pair1 : Initial number of reads ( /gfs/archive/jostins/microbiome/kneadata/demo/input/seq1.fastq ): 42473 > > 09/18/2020 10:08:37 AM - kneaddata.utilities - INFO: READ COUNT: raw pair2 : Initial number of reads ( /gfs/archive/jostins/microbiome/kneadata/demo/input/seq2.fastq ): 42473 > > 09/18/2020 10:08:37 AM - kneaddata.utilities - DEBUG: Checking input file to Trimmomatic : /gfs/archive/jostins/microbiome/kneadata/demo/input/seq1.fastq > > 09/18/2020 10:08:37 AM - kneaddata.utilities - DEBUG: Checking input file to Trimmomatic : /gfs/archive/jostins/microbiome/kneadata/demo/input/seq2.fastq > > 09/18/2020 10:08:37 AM - kneaddata.utilities - INFO: Running Trimmomatic … > > 09/18/2020 10:08:37 AM - kneaddata.utilities - INFO: Execute command: java -Xmx500m -jar /gfs/apps/bio/trimmomatic-0.35/trimmomatic-0.35.jar PE -threads 1 -phred33 /gfs/archive/jostins/microbiome/kneadata/demo/input/seq1.fastq /gfs/archive/jostins/microbiome/kneadata/demo/input/seq2.fastq /gfs/archive/jostins/microbiome/kneadata/demo/kneaddataOutputPairedEnd/seq1\_kneaddata.trimmed.1.fastq /gfs/archive/jostins/microbiome/kneadata/demo/kneaddataOutputPairedEnd/seq1\_kneaddata.trimmed.single.1.fastq /gfs/archive/jostins/microbiome/kneadata/demo/kneaddataOutputPairedEnd/seq1\_kneaddata.trimmed.2.fastq /gfs/archive/jostins/microbiome/kneadata/demo/kneaddataOutputPairedEnd/seq1\_kneaddata.trimmed.single.2.fastq ILLUMINACLIP:/gfs/devel/ljostins/python-venv-2.7.13/lib/python2.7/site-packages/kneaddata/adapters/NexteraPE-PE.fa:2:30:10:8:TRUE SLIDINGWINDOW:4:20 MINLEN:87 > > 09/18/2020 10:08:38 AM - kneaddata.utilities - DEBUG: TrimmomaticPE: Started with arguments: > > -threads 1 -phred33 /gfs/archive/jostins/microbiome/kneadata/demo/input/seq1.fastq /gfs/archive/jostins/microbiome/kneadata/demo/input/seq2.fastq /gfs/archive/jostins/microbiome/kneadata/demo/kneaddataOutputPairedEnd/seq1\_kneaddata.trimmed.1.fastq /gfs/archive/jostins/microbiome/kneadata/demo/kneaddataOutputPairedEnd/seq1\_kneaddata.trimmed.single.1.fastq /gfs/archive/jostins/microbiome/kneadata/demo/kneaddataOutputPairedEnd/seq1\_kneaddata.trimmed.2.fastq /gfs/archive/jostins/microbiome/kneadata/demo/kneaddataOutputPairedEnd/seq1\_kneaddata.trimmed.single.2.fastq ILLUMINACLIP:/gfs/devel/ljostins/python-venv-2.7.13/lib/python2.7/site-packages/kneaddata/adapters/NexteraPE-PE.fa:2:30:10:8:TRUE SLIDINGWINDOW:4:20 MINLEN:87 > > Using PrefixPair: ‘AGATGTGTATAAGAGACAG’ and ‘AGATGTGTATAAGAGACAG’ > > Using Long Clipping Sequence: ‘GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG’ > > Using Long Clipping Sequence: ‘TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG’ > > Using Long Clipping Sequence: ‘CTGTCTCTTATACACATCTCCGAGCCCACGAGAC’ > > Using Long Clipping Sequence: ‘CTGTCTCTTATACACATCTGACGCTGCCGACGA’ > > ILLUMINACLIP: Using 1 prefix pairs, 4 forward/reverse sequences, 0 forward only sequences, 0 reverse only sequences > > Input Read Pairs: 42473 Both Surviving: 35341 (83.21%) Forward Only Surviving: 5385 (12.68%) Reverse Only Surviving: 847 (1.99%) Dropped: 900 (2.12%) > > TrimmomaticPE: Completed successfully > > 09/18/2020 10:08:38 AM - kneaddata.utilities - DEBUG: Checking output file from Trimmomatic : /gfs/archive/jostins/microbiome/kneadata/demo/kneaddataOutputPairedEnd/seq1\_kneaddata.trimmed.1.fastq > > 09/18/2020 10:08:38 AM - kneaddata.utilities - DEBUG: Checking output file from Trimmomatic : /gfs/archive/jostins/microbiome/kneadata/demo/kneaddataOutputPairedEnd/seq1\_kneaddata.trimmed.single.1.fastq > > 09/18/2020 10:08:38 AM - kneaddata.utilities - DEBUG: Checking output file from Trimmomatic : /gfs/archive/jostins/microbiome/kneadata/demo/kneaddataOutputPairedEnd/seq1\_kneaddata.trimmed.2.fastq > > 09/18/2020 10:08:38 AM - kneaddata.utilities - DEBUG: Checking output file from Trimmomatic : /gfs/archive/jostins/microbiome/kneadata/demo/kneaddataOutputPairedEnd/seq1\_kneaddata.trimmed.single.2.fastq > > 09/18/2020 10:08:39 AM - kneaddata.utilities - INFO: READ COUNT: trimmed pair1 : Total reads after trimming ( /gfs/archive/jostins/microbiome/kneadata/demo/kneaddataOutputPairedEnd/seq1\_kneaddata.trimmed.1.fastq ): 35341 > > 09/18/2020 10:08:39 AM - kneaddata.utilities - INFO: READ COUNT: trimmed pair2 : Total reads after trimming ( /gfs/archive/jostins/microbiome/kneadata/demo/kneaddataOutputPairedEnd/seq1\_kneaddata.trimmed.2.fastq ): 35341 > > 09/18/2020 10:08:39 AM - kneaddata.utilities - INFO: READ COUNT: trimmed orphan1 : Total reads after trimming ( /gfs/archive/jostins/microbiome/kneadata/demo/kneaddataOutputPairedEnd/seq1\_kneaddata.trimmed.single.1.fastq ): 5385 > > 09/18/2020 10:08:39 AM - kneaddata.utilities - INFO: READ COUNT: trimmed orphan2 : Total reads after trimming ( /gfs/archive/jostins/microbiome/kneadata/demo/kneaddataOutputPairedEnd/seq1\_kneaddata.trimmed.single.2.fastq ): 847 > > 09/18/2020 10:08:40 AM - kneaddata.utilities - DEBUG: Checking input file to trf : /gfs/archive/jostins/microbiome/kneadata/demo/kneaddataOutputPairedEnd/seq1\_kneaddata.trimmed.1.fasta > > 09/18/2020 10:08:40 AM - kneaddata.utilities - INFO: Running trf … > > 09/18/2020 10:08:40 AM - kneaddata.utilities - INFO: Execute command: /gfs/archive/jostins/microbiome/software/trf/trf /gfs/archive/jostins/microbiome/kneadata/demo/kneaddataOutputPairedEnd/seq1\_kneaddata.trimmed.1.fasta 2 7 7 80 10 50 500 -h -ngs > > 09/18/2020 10:08:43 AM - kneaddata.utilities - DEBUG: 0 > > 09/18/2020 10:08:43 AM - kneaddata.utilities - DEBUG: Checking output file from trf : /gfs/archive/jostins/microbiome/kneadata/demo/kneaddataOutputPairedEnd/seq1\_kneaddata.trimmed.1.fasta.trf.parameters.2.7.7.80.10.50.500.dat > > 09/18/2020 10:08:43 AM - kneaddata.utilities - DEBUG: Checking input file to trf : /gfs/archive/jostins/microbiome/kneadata/demo/kneaddataOutputPairedEnd/seq1\_kneaddata.trimmed.2.fasta > > 09/18/2020 10:08:43 AM - kneaddata.utilities - INFO: Running trf … > > 09/18/2020 10:08:43 AM - kneaddata.utilities - INFO: Execute command: /gfs/archive/jostins/microbiome/software/trf/trf /gfs/archive/jostins/microbiome/kneadata/demo/kneaddataOutputPairedEnd/seq1\_kneaddata.trimmed.2.fasta 2 7 7 80 10 50 500 -h -ngs > > 09/18/2020 10:08:46 AM - kneaddata.utilities - DEBUG: 0 > > 09/18/2020 10:08:46 AM - kneaddata.utilities - DEBUG: Checking output file from trf : /gfs/archive/jostins/microbiome/kneadata/demo/kneaddataOutputPairedEnd/seq1\_kneaddata.trimmed.2.fasta.trf.parameters.2.7.7.80.10.50.500.dat > > 09/18/2020 10:08:47 AM - kneaddata.run - INFO: Total number of sequences with repeats removed from file ( /gfs/archive/jostins/microbiome/kneadata/demo/kneaddataOutputPairedEnd/seq1\_kneaddata.trimmed.1.fastq ): 0 > > 09/18/2020 10:08:47 AM - kneaddata.run - INFO: Total number of sequences with repeats removed from file ( /gfs/archive/jostins/microbiome/kneadata/demo/kneaddataOutputPairedEnd/seq1\_kneaddata.trimmed.2.fastq ): 0 > > 09/18/2020 10:08:47 AM - kneaddata.utilities - DEBUG: Checking input file to trf : /gfs/archive/jostins/microbiome/kneadata/demo/kneaddataOutputPairedEnd/seq1\_kneaddata.trimmed.single.1.fasta > > 09/18/2020 10:08:47 AM - kneaddata.utilities - INFO: Running trf … > > 09/18/2020 10:08:47 AM - kneaddata.utilities - INFO: Execute command: /gfs/archive/jostins/microbiome/software/trf/trf /gfs/archive/jostins/microbiome/kneadata/demo/kneaddataOutputPairedEnd/seq1\_kneaddata.trimmed.single.1.fasta 2 7 7 80 10 50 500 -h -ngs > > 09/18/2020 10:08:47 AM - kneaddata.utilities - DEBUG: 0 > > 09/18/2020 10:08:47 AM - kneaddata.utilities - DEBUG: Checking output file from trf : /gfs/archive/jostins/microbiome/kneadata/demo/kneaddataOutputPairedEnd/seq1\_kneaddata.trimmed.single.1.fasta.trf.parameters.2.7.7.80.10.50.500.dat > > 09/18/2020 10:08:47 AM - kneaddata.run - INFO: Total number of sequences with repeats removed from file ( /gfs/archive/jostins/microbiome/kneadata/demo/kneaddataOutputPairedEnd/seq1\_kneaddata.trimmed.single.1.fastq ): 0 > > 09/18/2020 10:08:47 AM - kneaddata.utilities - DEBUG: Checking input file to trf : /gfs/archive/jostins/microbiome/kneadata/demo/kneaddataOutputPairedEnd/seq1\_kneaddata.trimmed.single.2.fasta > > 09/18/2020 10:08:47 AM - kneaddata.utilities - INFO: Running trf … > > 09/18/2020 10:08:47 AM - kneaddata.utilities - INFO: Execute command: /gfs/archive/jostins/microbiome/software/trf/trf /gfs/archive/jostins/microbiome/kneadata/demo/kneaddataOutputPairedEnd/seq1\_kneaddata.trimmed.single.2.fasta 2 7 7 80 10 50 500 -h -ngs > > 09/18/2020 10:08:48 AM - kneaddata.utilities - DEBUG: 0 > > 09/18/2020 10:08:48 AM - kneaddata.utilities - DEBUG: Checking output file from trf : /gfs/archive/jostins/microbiome/kneadata/demo/kneaddataOutputPairedEnd/seq1\_kneaddata.trimmed.single.2.fasta.trf.parameters.2.7.7.80.10.50.500.dat > > 09/18/2020 10:08:48 AM - kneaddata.run - INFO: Total number of sequences with repeats removed from file ( /gfs/archive/jostins/microbiome/kneadata/demo/kneaddataOutputPairedEnd/seq1\_kneaddata.trimmed.single.2.fastq ): 1 > > 09/18/2020 10:08:48 AM - kneaddata.run - INFO: Decontaminating … > > 09/18/2020 10:08:48 AM - kneaddata.utilities - DEBUG: Checking input file to bowtie2 : /gfs/archive/jostins/microbiome/kneadata/demo/kneaddataOutputPairedEnd/seq1\_kneaddata.repeats.removed.1.fastq > > 09/18/2020 10:08:48 AM - kneaddata.utilities - DEBUG: Checking input file to bowtie2 : /gfs/archive/jostins/microbiome/kneadata/demo/kneaddataOutputPairedEnd/seq1\_kneaddata.repeats.removed.2.fastq > > 09/18/2020 10:08:48 AM - kneaddata.utilities - DEBUG: Checking input file to bowtie2 : /gfs/archive/jostins/microbiome/kneadata/demo/kneaddataOutputPairedEnd/seq1\_kneaddata.repeats.removed.unmatched.1.fastq > > 09/18/2020 10:08:48 AM - kneaddata.utilities - DEBUG: Checking input file to bowtie2 : /gfs/archive/jostins/microbiome/kneadata/demo/kneaddataOutputPairedEnd/seq1\_kneaddata.repeats.removed.unmatched.2.fastq > > 09/18/2020 10:08:48 AM - kneaddata.utilities - INFO: Running bowtie2 … > > 09/18/2020 10:08:48 AM - kneaddata.utilities - INFO: Execute command: kneaddata\_bowtie2\_discordant\_pairs --bowtie2 /gfs/apps/bio/bowtie2-2.3.0/bowtie2 --threads 1 -x /gfs/archive/jostins/microbiome/kneadata/demo/input/demo\_db --bowtie2-options “–very-sensitive --phred33” -1 /gfs/archive/jostins/microbiome/kneadata/demo/kneaddataOutputPairedEnd/seq1\_kneaddata.repeats.removed.1.fastq -2 /gfs/archive/jostins/microbiome/kneadata/demo/kneaddataOutputPairedEnd/seq1\_kneaddata.repeats.removed.2.fastq --un-pair /gfs/archive/jostins/microbiome/kneadata/demo/kneaddataOutputPairedEnd/seq1\_kneaddata\_demo\_db\_bowtie2\_paired\_clean\_%.fastq --al-pair /gfs/archive/jostins/microbiome/kneadata/demo/kneaddataOutputPairedEnd/seq1\_kneaddata\_demo\_db\_bowtie2\_paired\_contam\_%.fastq -U /gfs/archive/jostins/microbiome/kneadata/demo/kneaddataOutputPairedEnd/seq1\_kneaddata.repeats.removed.unmatched.1.fastq,/gfs/archive/jostins/microbiome/kneadata/demo/kneaddataOutputPairedEnd/seq1\_kneaddata.repeats.removed.unmatched.2.fastq --un-single /gfs/archive/jostins/microbiome/kneadata/demo/kneaddataOutputPairedEnd/seq1\_kneaddata\_demo\_db\_bowtie2\_unmatched\_%_clean.fastq --al-single /gfs/archive/jostins/microbiome/kneadata/demo/kneaddataOutputPairedEnd/seq1\_kneaddata\_demo\_db\_bowtie2\_unmatched_%\_contam.fastq -S /dev/null > > 09/18/2020 10:08:49 AM - kneaddata.utilities - DEBUG: 35341 reads; of these: > > 35341 (100.00%) were unpaired; of these: > > 35341 (100.00%) aligned 0 times > > 0 (0.00%) aligned exactly 1 time > > 0 (0.00%) aligned \>1 times > > 0.00% overall alignment rate > > pair1\_aligned : 0 > > pair2\_aligned : 0 > > orphan1\_unaligned : 35341 > > orphan2\_unaligned : 0 > > orphan2\_aligned : 0 > > pair2\_unaligned : 0 > > pair1\_unaligned : 0 > > orphan1\_aligned : 0 > > 09/18/2020 10:08:49 AM - kneaddata.utilities - DEBUG: Checking output file from bowtie2 : /gfs/archive/jostins/microbiome/kneadata/demo/kneaddataOutputPairedEnd/seq1\_kneaddata\_demo\_db\_bowtie2\_paired\_clean\_1.fastq > > 09/18/2020 10:08:49 AM - kneaddata.utilities - DEBUG: Checking output file from bowtie2 : /gfs/archive/jostins/microbiome/kneadata/demo/kneaddataOutputPairedEnd/seq1\_kneaddata\_demo\_db\_bowtie2\_paired\_clean\_2.fastq > > 09/18/2020 10:08:49 AM - kneaddata.run - INFO: Total contaminate sequences in file ( /gfs/archive/jostins/microbiome/kneadata/demo/kneaddataOutputPairedEnd/seq1\_kneaddata\_demo\_db\_bowtie2\_paired\_contam\_1.fastq ) : 0 > > 09/18/2020 10:08:49 AM - kneaddata.run - INFO: Total contaminate sequences in file ( /gfs/archive/jostins/microbiome/kneadata/demo/kneaddataOutputPairedEnd/seq1\_kneaddata\_demo\_db\_bowtie2\_paired\_contam\_2.fastq ) : 0 > > 09/18/2020 10:08:49 AM - kneaddata.run - INFO: Total contaminate sequences in file ( /gfs/archive/jostins/microbiome/kneadata/demo/kneaddataOutputPairedEnd/seq1\_kneaddata\_demo\_db\_bowtie2\_unmatched\_1\_contam.fastq ) : 0 > > 09/18/2020 10:08:49 AM - kneaddata.run - INFO: Total contaminate sequences in file ( /gfs/archive/jostins/microbiome/kneadata/demo/kneaddataOutputPairedEnd/seq1\_kneaddata\_demo\_db\_bowtie2\_unmatched\_2\_contam.fastq ) : 0 > > 09/18/2020 10:08:49 AM - kneaddata.utilities - INFO: READ COUNT: decontaminated demo\_db pair1 : Total reads after removing those found in reference database ( /gfs/archive/jostins/microbiome/kneadata/demo/kneaddataOutputPairedEnd/seq1\_kneaddata\_demo\_db\_bowtie2\_paired\_clean\_1.fastq ): 0 > > 09/18/2020 10:08:49 AM - kneaddata.utilities - INFO: READ COUNT: decontaminated demo\_db pair2 : Total reads after removing those found in reference database ( /gfs/archive/jostins/microbiome/kneadata/demo/kneaddataOutputPairedEnd/seq1\_kneaddata\_demo\_db\_bowtie2\_paired\_clean\_2.fastq ): 0 > > 09/18/2020 10:08:49 AM - kneaddata.utilities - INFO: READ COUNT: final pair1 : Total reads after merging results from multiple databases ( /gfs/archive/jostins/microbiome/kneadata/demo/kneaddataOutputPairedEnd/seq1\_kneaddata\_paired\_1.fastq ): 0 > > 09/18/2020 10:08:49 AM - kneaddata.utilities - INFO: READ COUNT: final pair2 : Total reads after merging results from multiple databases ( /gfs/archive/jostins/microbiome/kneadata/demo/kneaddataOutputPairedEnd/seq1\_kneaddata\_paired\_2.fastq ): 0 > > 09/18/2020 10:08:49 AM - kneaddata.utilities - WARNING: Unable to remove file: /gfs/archive/jostins/microbiome/kneadata/demo/kneaddataOutputPairedEnd/seq1\_kneaddata\_demo\_db\_bowtie2\_paired\_clean\_1.fastq > > 09/18/2020 10:08:49 AM - kneaddata.utilities - WARNING: Unable to remove file: /gfs/archive/jostins/microbiome/kneadata/demo/kneaddataOutputPairedEnd/seq1\_kneaddata\_demo\_db\_bowtie2\_paired\_clean\_2.fastq > > 09/18/2020 10:08:49 AM - kneaddata.utilities - INFO: READ COUNT: decontaminated demo\_db orphan1 : Total reads after removing those found in reference database ( /gfs/archive/jostins/microbiome/kneadata/demo/kneaddataOutputPairedEnd/seq1\_kneaddata\_demo\_db\_bowtie2\_unmatched\_1\_clean.fastq ): 35341 > > 09/18/2020 10:08:49 AM - kneaddata.utilities - INFO: READ COUNT: final orphan1 : Total reads after merging results from multiple databases ( /gfs/archive/jostins/microbiome/kneadata/demo/kneaddataOutputPairedEnd/seq1\_kneaddata\_unmatched\_1.fastq ): 35341 > > 09/18/2020 10:08:49 AM - kneaddata.utilities - WARNING: Unable to remove file: /gfs/archive/jostins/microbiome/kneadata/demo/kneaddataOutputPairedEnd/seq1\_kneaddata\_demo\_db\_bowtie2\_unmatched\_1\_clean.fastq > > 09/18/2020 10:08:49 AM - kneaddata.utilities - INFO: READ COUNT: decontaminated demo\_db orphan2 : Total reads after removing those found in reference database ( /gfs/archive/jostins/microbiome/kneadata/demo/kneaddataOutputPairedEnd/seq1\_kneaddata\_demo\_db\_bowtie2\_unmatched\_2\_clean.fastq ): 0 > > 09/18/2020 10:08:49 AM - kneaddata.utilities - INFO: READ COUNT: final orphan2 : Total reads after merging results from multiple databases ( /gfs/archive/jostins/microbiome/kneadata/demo/kneaddataOutputPairedEnd/seq1\_kneaddata\_unmatched\_2.fastq ): 0 > > 09/18/2020 10:08:49 AM - kneaddata.utilities - WARNING: Unable to remove file: /gfs/archive/jostins/microbiome/kneadata/demo/kneaddataOutputPairedEnd/seq1\_kneaddata\_demo\_db\_bowtie2\_unmatched\_2\_clean.fastq > > 09/18/2020 10:08:49 AM - kneaddata.knead\_data - INFO: > > Final output files created: > > /gfs/archive/jostins/microbiome/kneadata/demo/kneaddataOutputPairedEnd/seq1\_kneaddata\_paired\_1.fastq > > /gfs/archive/jostins/microbiome/kneadata/demo/kneaddataOutputPairedEnd/seq1\_kneaddata\_paired\_2.fastq > > /gfs/archive/jostins/microbiome/kneadata/demo/kneaddataOutputPairedEnd/seq1\_kneaddata\_unmatched\_1.fastq > > /gfs/archive/jostins/microbiome/kneadata/demo/kneaddataOutputPairedEnd/seq1\_kneaddata\_unmatched\_2.fastq ---
**Author:** ![lowk](https://yyz1.discourse-cdn.com/flex027/user_avatar/forum.biobakery.org/lowk/32/392_2.png) [@lowk](https://forum.biobakery.org/u/lowk)\ **Post date:** [September 18, 2020, 11:46am UTC](https://forum.biobakery.org/t/problem-with-paired-end-demo-on-new-install/1004/4 "2020-09-18T11:46:31Z")
Oh, I think the issue here might be with bowtie2. If update from 2.3.0 to 2.4.1 it seems to work: > bash$ kneaddata --input input/seq1.fastq --input input/seq2.fastq --reference-db input/demo\_db --output kneaddataOutputPairedEnd --trf …/…/software/trf/ --bowtie2 /gfs/archive/jostins/microbiome/software/bowtie2-2.4.1-linux-x86\_64/ > $ kneaddata --input input/seq1.fastq --input input/seq2.fastq --reference-db input/demo\_db --output kneaddataOutputPairedEnd --trf …/…/software/trf/ --bowtie2 /gfs/archive/jostins/microbiome/software/bowtie2-2.4.1-linux-x86\_64/ > > Initial number of reads ( /gfs/archive/jostins/microbiome/kneadata/demo/input/seq1.fastq ): 42473 > > Initial number of reads ( /gfs/archive/jostins/microbiome/kneadata/demo/input/seq2.fastq ): 42473 > > Running Trimmomatic … > > Total reads after trimming ( /gfs/archive/jostins/microbiome/kneadata/demo/kneaddataOutputPairedEnd/seq1\_kneaddata.trimmed.1.fastq ): 35341 > > Total reads after trimming ( /gfs/archive/jostins/microbiome/kneadata/demo/kneaddataOutputPairedEnd/seq1\_kneaddata.trimmed.2.fastq ): 35341 > > Total reads after trimming ( /gfs/archive/jostins/microbiome/kneadata/demo/kneaddataOutputPairedEnd/seq1\_kneaddata.trimmed.single.1.fastq ): 5385 > > Total reads after trimming ( /gfs/archive/jostins/microbiome/kneadata/demo/kneaddataOutputPairedEnd/seq1\_kneaddata.trimmed.single.2.fastq ): 847 > > Running trf … > > Running trf … > > Running trf … > > Running trf … > > Decontaminating … > > Running bowtie2 … > > Total reads after removing those found in reference database ( /gfs/archive/jostins/microbiome/kneadata/demo/kneaddataOutputPairedEnd/seq1\_kneaddata\_demo\_db\_bowtie2\_paired\_clean\_1.fastq ): 35341 > > Total reads after removing those found in reference database ( /gfs/archive/jostins/microbiome/kneadata/demo/kneaddataOutputPairedEnd/seq1\_kneaddata\_demo\_db\_bowtie2\_paired\_clean\_2.fastq ): 35341 > > Total reads after merging results from multiple databases ( /gfs/archive/jostins/microbiome/kneadata/demo/kneaddataOutputPairedEnd/seq1\_kneaddata\_paired\_1.fastq ): 35341 > > Total reads after merging results from multiple databases ( /gfs/archive/jostins/microbiome/kneadata/demo/kneaddataOutputPairedEnd/seq1\_kneaddata\_paired\_2.fastq ): 35341 > > Total reads after removing those found in reference database ( /gfs/archive/jostins/microbiome/kneadata/demo/kneaddataOutputPairedEnd/seq1\_kneaddata\_demo\_db\_bowtie2\_unmatched\_1\_clean.fastq ): 5385 > > Total reads after merging results from multiple databases ( /gfs/archive/jostins/microbiome/kneadata/demo/kneaddataOutputPairedEnd/seq1\_kneaddata\_unmatched\_1.fastq ): 5385 > > Total reads after removing those found in reference database ( /gfs/archive/jostins/microbiome/kneadata/demo/kneaddataOutputPairedEnd/seq1\_kneaddata\_demo\_db\_bowtie2\_unmatched\_2\_clean.fastq ): 846 > > Total reads after merging results from multiple databases ( /gfs/archive/jostins/microbiome/kneadata/demo/kneaddataOutputPairedEnd/seq1\_kneaddata\_unmatched\_2.fastq ): 846 > > Final output files created: > > /gfs/archive/jostins/microbiome/kneadata/demo/kneaddataOutputPairedEnd/seq1\_kneaddata\_paired\_1.fastq > > /gfs/archive/jostins/microbiome/kneadata/demo/kneaddataOutputPairedEnd/seq1\_kneaddata\_paired\_2.fastq > > /gfs/archive/jostins/microbiome/kneadata/demo/kneaddataOutputPairedEnd/seq1\_kneaddata\_unmatched\_1.fastq > > /gfs/archive/jostins/microbiome/kneadata/demo/kneaddataOutputPairedEnd/seq1\_kneaddata\_unmatched\_2.fastq > bash$ wc kneaddataOutputPairedEnd/\* > 0 0 0 kneaddataOutputPairedEnd/seq1\_kneaddata\_demo\_db\_bowtie2\_paired\_contam\_1.fastq > 0 0 0 kneaddataOutputPairedEnd/seq1\_kneaddata\_demo\_db\_bowtie2\_paired\_contam\_2.fastq > 0 0 0 kneaddataOutputPairedEnd/seq1\_kneaddata\_demo\_db\_bowtie2\_unmatched\_1\_contam.fastq > 0 0 0 kneaddataOutputPairedEnd/seq1\_kneaddata\_demo\_db\_bowtie2\_unmatched\_2\_contam.fastq > 138 1354 17872 kneaddataOutputPairedEnd/seq1\_kneaddata.log > 141364 141364 13333610 kneaddataOutputPairedEnd/seq1\_kneaddata\_paired\_1.fastq > 141364 141364 13302786 kneaddataOutputPairedEnd/seq1\_kneaddata\_paired\_2.fastq > 141364 141364 13333610 kneaddataOutputPairedEnd/seq1\_kneaddata.repeats.removed.1.fastq > 141364 141364 13302786 kneaddataOutputPairedEnd/seq1\_kneaddata.repeats.removed.2.fastq > 21540 21540 1899203 kneaddataOutputPairedEnd/seq1\_kneaddata.repeats.removed.unmatched.1.fastq > 3384 3384 289698 kneaddataOutputPairedEnd/seq1\_kneaddata.repeats.removed.unmatched.2.fastq > 141364 141364 13333610 kneaddataOutputPairedEnd/seq1\_kneaddata.trimmed.1.fastq > 141364 141364 13302786 kneaddataOutputPairedEnd/seq1\_kneaddata.trimmed.2.fastq > 21540 21540 1899203 kneaddataOutputPairedEnd/seq1\_kneaddata.trimmed.single.1.fastq > 3388 3388 290085 kneaddataOutputPairedEnd/seq1\_kneaddata.trimmed.single.2.fastq > 21540 21540 1899203 kneaddataOutputPairedEnd/seq1\_kneaddata\_unmatched\_1.fastq > 3384 3384 289698 kneaddataOutputPairedEnd/seq1\_kneaddata\_unmatched\_2.fastq > 923098 924314 86494150 total ---
**Author:** ![lauren.j.mciver](https://avatars.discourse-cdn.com/v4/letter/l/f05b48/32.png) [@lauren.j.mciver](https://forum.biobakery.org/u/lauren.j.mciver)\ **Post date:** [September 18, 2020, 7:55pm UTC](https://forum.biobakery.org/t/problem-with-paired-end-demo-on-new-install/1004/5 "2020-09-18T19:55:12Z")
Hi - That is great you have it figured out! Thanks for the follow up post. We will make a note of the bowtie2 version on our end. Thank you, Lauren ---
**Author:** ![EmilyB](https://avatars.discourse-cdn.com/v4/letter/e/5daacb/32.png) [@EmilyB](https://forum.biobakery.org/u/EmilyB)\ **Post date:** [November 26, 2021, 11:29pm UTC](https://forum.biobakery.org/t/problem-with-paired-end-demo-on-new-install/1004/6 "2021-11-26T23:29:48Z")
Hi Lauren - I am having the same error but with bowtie2 version 2.4.2. Any thoughts on what might be causing it? ---
**Author:** ![wang](https://yyz1.discourse-cdn.com/flex027/user_avatar/forum.biobakery.org/wang/32/1152_2.png) [@wang](https://forum.biobakery.org/u/wang)\ **Post date:** [December 13, 2021, 3:15am UTC](https://forum.biobakery.org/t/problem-with-paired-end-demo-on-new-install/1004/7 "2021-12-13T03:15:26Z")
Hi, @EmilyB @lauren.j.mciver ,I am having the same error but with bowtie2 version 2.4.4 and version 2.3.5, have you solved this problem? ---
**Author:** ![EmilyB](https://avatars.discourse-cdn.com/v4/letter/e/5daacb/32.png) [@EmilyB](https://forum.biobakery.org/u/EmilyB)\ **Post date:** [December 29, 2021, 6:57pm UTC](https://forum.biobakery.org/t/problem-with-paired-end-demo-on-new-install/1004/8 "2021-12-29T18:57:28Z")
No, I haven’t. I will try stating the specific bowtie2 version in kneaddata (like above) ---
**Author:** ![JenS](https://avatars.discourse-cdn.com/v4/letter/j/d2c977/32.png) [@JenS](https://forum.biobakery.org/u/JenS)\ **Post date:** [March 8, 2022, 1:47pm UTC](https://forum.biobakery.org/t/problem-with-paired-end-demo-on-new-install/1004/9 "2022-03-08T13:47:07Z")
I have this same error. Did anyone solve this? ---
**Author:** ![wang](https://yyz1.discourse-cdn.com/flex027/user_avatar/forum.biobakery.org/wang/32/1152_2.png) [@wang](https://forum.biobakery.org/u/wang)\ **Post date:** [March 19, 2022, 8:25am UTC](https://forum.biobakery.org/t/problem-with-paired-end-demo-on-new-install/1004/11 "2022-03-19T08:25:38Z")
Yes, the reason is that kneaddata didn’t recognize the sequence label, I replaced the sequence with other labels, and then it solved. ---
**Author:** ![Rabindra](https://avatars.discourse-cdn.com/v4/letter/r/8e8cbc/32.png) [@Rabindra](https://forum.biobakery.org/u/Rabindra)\ **Post date:** [April 7, 2022, 11:12am UTC](https://forum.biobakery.org/t/problem-with-paired-end-demo-on-new-install/1004/12 "2022-04-07T11:12:38Z")
Hi Wang, I have encountered the same problem. How did you replace the sequence labels? Can you provide the command? Thanks, Rabindra ---
**Author:** ![Rabindra](https://avatars.discourse-cdn.com/v4/letter/r/8e8cbc/32.png) [@Rabindra](https://forum.biobakery.org/u/Rabindra)\ **Post date:** [April 9, 2022, 8:49am UTC](https://forum.biobakery.org/t/problem-with-paired-end-demo-on-new-install/1004/13 "2022-04-09T08:49:53Z")
Hi @lauren.j.mciver and @wang I am having similar issues. Even replacing sequence labels and changing bowtie version to 2.4.2 does not seem to work. Thanks, Rabidra ---
**Author:** ![fquerdasi](https://yyz1.discourse-cdn.com/flex027/user_avatar/forum.biobakery.org/fquerdasi/32/927_2.png) [@fquerdasi](https://forum.biobakery.org/u/fquerdasi)\ **Post date:** [August 28, 2023, 10:36pm UTC](https://forum.biobakery.org/t/problem-with-paired-end-demo-on-new-install/1004/14 "2023-08-28T22:36:05Z")
> [@lowk](#): > > 2.4.1 Hi @Rabindra, Did you end up figuring out a different solution? I’m having the same issue and replacing sequence labels has not worked for me. I’ve yet to try changing the bowtie2 version but I’d also appreciate any other suggestions in case that doesn’t work for me. Thanks! Fran ---
**Author:** ![fquerdasi](https://yyz1.discourse-cdn.com/flex027/user_avatar/forum.biobakery.org/fquerdasi/32/927_2.png) [@fquerdasi](https://forum.biobakery.org/u/fquerdasi)\ **Post date:** [August 31, 2023, 1:30am UTC](https://forum.biobakery.org/t/problem-with-paired-end-demo-on-new-install/1004/15 "2023-08-31T01:30:29Z")
Yup, still have the issue even with bowtie2 version 2.4.1. Any other suggestions would be greatly appreciated. ---
**Author:** ![vidya\_patil](https://yyz1.discourse-cdn.com/flex027/user_avatar/forum.biobakery.org/vidya_patil/32/2812_2.png) [@vidya\_patil](https://forum.biobakery.org/u/vidya_patil)\ **Post date:** [April 8, 2024, 4:17pm UTC](https://forum.biobakery.org/t/problem-with-paired-end-demo-on-new-install/1004/16 "2024-04-08T16:17:32Z")
Hi Fran, I am also facing the same issue with the kneaddata v0.12.0 version, I tried modifying the read identification however did not work. kneaddata v0.10.0 is not working for me. Did you find the solution? Thank you in advance. Vidya ---
**Author:** ![eo24a](https://avatars.discourse-cdn.com/v4/letter/e/b9bd4f/32.png) [@eo24a](https://forum.biobakery.org/u/eo24a)\ **Post date:** [October 4, 2024, 12:49am UTC](https://forum.biobakery.org/t/problem-with-paired-end-demo-on-new-install/1004/17 "2024-10-04T00:49:24Z")
I am having the same issue here. Just downloaded kneaddata v0.12.0 with pip install and bowtie2 v2.4.2, the program runs with no error or warning but the paired fastq files are 0 bytes. It seems like it did not delete any contamination from the reads. Has anyone solved this issue and how?