(r12) Barrick Lab :: ProtocolsFlexbarCommands

---+ Using Flexbar program to remove adapter sequences from NGS reads

---++ Installing Flexbar on Mac.

   1 Go to [[http://sourceforge.net/projects/flexbar/files/][Flexbar home page]] select the newest version (2.5 as of 8-12-14).
   1 Download _macosx.tgz file.
   1 Move downloaded file to more permanent location (ie source)
   1 Extract downloaded file.
   1 Extracted folder should contain 3 things:
      1 README.txt
      1 flexbar
      1 libtbb.dylib
   1 copy flexbar to your path, or add extracted folder to path.
      * suggest copying to $HOME/local/bin
   1 modify your .profile ($HOME/.profile) to include the following if not included already
      1 export PATH=$HOME/local/bin:$PATH
      1 export DYLD_LIBRARY_PATH="PATH-TO-YOUR-EXTRACTED-FOLDER":$DYLD_LIBRARY_PATH
         * replace everything including the quotation marks with the actual location of your extracted folder.
   1 open new terminal and flexbar should be working
 

---++ Installing Flexbar on TACC.

   * *Currently Flexbar is installed on TACC under the BioITeam, but is only accessable as a job (que or idev) on lonestar. Stampede requires running on head node (bad idea) or copying it to a local directory in your path. Below are instructions for installing it yourself instead.*
   1 Go to [[http://sourceforge.net/projects/flexbar/files/][Flexbar home page]] select the newest version (2.5 as of 8-12-14).
   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.

If you try installing from source, you may need to switch to the gcc compiler (module swap intel gcc)

---++ Command line usage for removal of adapter sequences

*The following does not apply to standard illumina sequencing libraries including MiSeq runs.* DNAzyme project (this involved constant non-informative regions flanking each side of a library) revealed unexpected flexbar behavior. Specifically both the left and right ends of a single sequence can not be modified in a single pass. All "adapter" sequences are aligned to each read at once, and the sequence with the best alignment is acted on. This means that in a single pass only the left or right adapter can be trimmed. To avoid this recommend that 2 passes are performed: left, then right.
Generic command for performing maximal (aggressive) trimming. Replace everything between "" with appropriate names, and delete the "" marks:

<code>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 </code>

Example command:

<code>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</code>

For a less aggressive command, remove <code>-ao 1</code>.

---++ Choice of adaptor sequence file

For most data sets analyzed in the lab, the Illumina Truseq adaptors file is the correct one to use (attached file: <code>illumina_truseq.fasta</code>).

---++ 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 help and options, type <code>flexbar -h</code>

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
fasta	illumina_nextera.fasta	r1	manage	0.2 K	2014-04-25 - 03:45	JeffreyBarrick
fasta	illumina_truseq.fasta	r1	manage	0.1 K	2014-04-18 - 17:22	JeffreyBarrick
fasta	new_adaptors.fasta	r1	manage	0.3 K	2013-05-21 - 16:47	DanielDeatherage	fasta_file_of_adapter_sequences 8bp barcode sequences

Barrick Lab > ProtocolList > ProtocolsFlexbarCommands

Contributors to this topic

DanielDeatherage, JeffreyBarrick

Topic revision: r12 - 2014-08-12 - 20:33:07 - Main.DanielDeatherage