---+ 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 [[http://sourceforge.net/projects/flexbar/files/][Flexbar home page]] select the newest version (2.31 as of 1-16-13). 1 Right click *_linux64.tgz and select 'copy link location'. 1 Log onto TACC 1 cd $WORK/src 1 wget "paste link location" * wget http://sourceforge.net/projects/flexbar/files/2.31/flexbar_v2.31_linux64.tgz/download 1 tar xvzf flexbar*.tgz 1 cd "new folder" * cd flexbar_v2.31_linux64 1 cp flexbar $HOME/local/bin 1 vi $HOME/.profile_user * Add the following if not already present: 1 export PATH=$HOME/local/bin:$PATH 1 export LD_LIBRARY_PATH=$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 1 logout 1 Log back onto TACC 1 flexbar -h 1 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) 1 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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Topic revision: r4 - 2013-02-01 - DanielDeatherage