Using Flexbar program to remove adapter sequences from NGS reads
Installing Flexbar (notes specific to TACC, can be updated for other systems)
- Go to Flexbar home page select the newest version (2.31 as of 1-16-13).
- Right click *_linux64.tgz and select 'copy link location'.
- Log onto TACC
- cd $WORK/src
- wget "paste link location"
- tar xvzf flexbar*.tgz
- cd "new folder"
- cp flexbar $HOME/local/bin
- vi $HOME/.profile_user
- Add the following if not already present:
- export PATH=$HOME/local/bin:$PATH
- export LD_LIBRARY_PATH=$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
- logout
- Log back onto TACC
- flexbar -h
- 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
Generic command for performing maximal (aggressive) trimming. 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 aggressive command, remove
-ao 1
.
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:
illumina_truseq.fasta
).
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
flexbar -h
Contributors to this topic
DanielDeatherage, JeffreyBarrick
Topic revision: r10 - 2014-04-25 - 03:45:32 - Main.JeffreyBarrick