(r7) Barrick Lab :: ProtocolsFlexbarCommands

---+ 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 -u 101 -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.|
|-u|Number|Number of N's allowed in final sequence. By default 0 Ns allowed. Breseq handles Ns therefore reads should/can be retained.|
|-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
Attachments
Topic attachments
I	Attachment	History	Action	Size	Date	Who	Comment
fasta	gsaf_illumina_adapters.fasta	r2 r1	manage	0.4 K	2013-03-11 - 17:29	JeffreyBarrick
Barrick Lab > ProtocolList > ProtocolsFlexbarCommands
Contributors to this topic
DanielDeatherage, JeffreyBarrick
Topic revision: r7 - 2013-04-29 - 19:06:43 - Main.DanielDeatherage