Using Flexbar program to remove adapter sequences from NGS reads

Installing Flexbar (notes specific to TACC, can be updated for other systems)

  1. Go to Flexbar home page select the newest version (2.31 as of 1-16-13).
  2. Right click *_linux64.tgz and select 'copy link location'.
  3. Log onto TACC
  4. cd $WORK/src
  5. wget "paste link location"
  6. tar xvzf flexbar*.tgz
  7. cd "new folder"
    • cd flexbar_v2.31_linux64
  8. cp flexbar $HOME/local/bin
  9. vi $HOME/.profile_user
    • Add the following if not already present:
      1. export PATH=$HOME/local/bin:$PATH
      2. export LD_LIBRARY_PATH=$WORK/src/flexbar_v2.31_linux64:$LD_LIBRARY_PATH
        • optionally, can move flexbar to any location in your path, and can move libtbb.so.2 to any location in LD_LIBRARY_PATH
  10. logout
  11. Log back onto TACC
  12. flexbar -h
  13. If the help manual appears flexbar should be ready to use. If you get an error message see below, and check that $PATH and $LD_LIBRARY_PATH include the locations of the relevant files.

Notes for TACC

  1. Lonestar does not allow intel/11.1 compiler. Doing so results in 2 error messages:
    • flexbar: /usr/lib64/libstdc++.so.6: version `GLIBCXX_3.4.11' not found (required by flexbar)
    • flexbar: /usr/lib64/libstdc++.so.6: version `GLIBCXX_3.4.9' not found (required by flexbar)
  2. Fix by adding following command to .profile file located in $HOME:
    • module swap intel gcc

Command line usage for removal of adapter sequences

Generic conservative command. Replace everything between "" with appropriate names, and delete the "" marks:

flexbar -t "New_file_name" -r "read_1_file_name" -p "read_2_file_name" -f fastq -a "fasta_file_of_adapter_sequences" -ao 1

Example command:

flexbar -t DED81 -r 02_Downloads/Sample_DED81_L004_R1.cat.fastq -p 02_Downloads/Sample_DED81_L004_R2.cat.fastq -f fastq -a 02_trimmed_Downloads/adapter_seq.fasta -ao 1

For a less conservative command, remove -ao 1

Flag explanations

Flag Text to follow What flag means Reason
-t New_file_name Name of output file. Dictate what your output file is to be named. Suggest something different than input to avoid overwriting untrimmed.
-r R1_source_file_name Name of Read1 sequencing file. File to remove adapters from.
-p R2_source_file_name Name of Read2 sequencing file.(Optional: can do each file separately). File to remove adapters from.
-f Format Format of reads. Most commonly will be fasta or fastq.
-a Adapter_sequence_file.fasta Fasta file with full adapter sequences, degenerate bases allowed. What sequence is to be removed.
-ao Number Number of bases of overlap between read and adapter This number equals the minimum number of bp to be removed.
-at Number Number of mismatches and indels per 10bp of adapter sequence allowed This accounts for sequencing/PCR errors changing adapter sequence. Default = 3, increasing this number increases false positive rate, and decreases false negative rate.

Additional Information

For additional information, type flexbar -h

-- Main.DanielDeatherage - 16 Jan 2013

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Contributors to this topic Edit topic DanielDeatherage, JeffreyBarrick
Topic revision: r5 - 2013-03-08 - 17:11:33 - Main.JeffreyBarrick
 
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