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VDJFasta
VDJFasta was developed for the analysis of antibody repertoires by high-throughput sequencing. It was was published as part of the methods in
"Precise determination of the diversity of a combinatorial antibody library gives insight into the human immunoglobulin repertoire" - Glanville, Zhai, Berka et all 2009 PNAS
http://www.pnas.org/content/early/2009/10/28/0909775106.short
The software was released as part of the methods of that publication, and is made available to the community for non-commercial purposes. The software is provided "as is," with no implied support. For those interested in contributing code to the package, the author can be reached at Jacob <dot> Glanville <at> pfizer <dot> com.
Dependencies
VDJFasta requires NCBI's blast toolkit, HMMER's hmm toolkit, and Perl.
Installation
NOTE: The current installation is easy, but crude. Any assistance in grooming the package to improve the installation experience would be greatly appreciated
The package can be unpacked as
$ tar -xzvf vdjfasta-1.0.tgz
This will create the following directory structure
vdjfasta/bin/ <-- bin directory with executable perl scripts
/db/ <-- blast and hmm reference databases
/lib/ <-- perl module libraries
/test/ <-- example sequences to test the software
/README.txt <-- this readme file
Once unpacked, a number of paths will need to be set in the constructor of vdjfasta/lib/VDJFasta.pm Adjust the paths to your current Blast and HMMER installations:
$self->{blast} = "/tools/apps/NCBI/blast-2.2.17/bin/blastall";
$self->{hmmsearch} = "/tools/apps/HMMER/hmmsearch";
$self->{hmmalign} = "/tools/apps/HMMER/hmmalign";
and the the current location of your vdjfasta installation:
$self->{dnaVsegdb} = "/tools/vdjfasta/db/imgt.VDJ.dna.nr.fa";
$self->{dnaJsegdb} = "/tools/vdjfasta/db/imgt.J.dna.fa";
$self->{dnaDsegdb} = "/tools/vdjfasta/db/imgt.HD.dna.nr.fa";
$self->{dnaCsegdb} = "/tools/vdjfasta/db/imgt.CH1.dna.nr.fa";
$self->{VhVkHMM} = "/tools/vdjfasta/db/Vh-linker-Vk.hmm";
Finally, add vdjfasta/bin to your path.
Description
The scripts available in the package are all located in vdjfasta/bin. Terse usage descriptions are provided if the scripts are executed without arguments.
bin: scripts available in the package
fasta-vdj-pipeline.pl complete analysis of antibody nucleotide sequences
fasta-dna2ig.pl identify and translate antibody coding frames
fasta-getGermline.pl identify segment composition
fasta-scFv-align.pl align antibody sequences
fasta-vdj-sim.pl perform antibody vdj simulations
msa-restack.pl perform CDR-restacking on scFv output alignment
Output
The fasta-vdj-pipeline.pl carries dna sequences from initial screening through full segment characterization, translation, and amino acid identification of CDRs. An example of input and output is shown below:
Input would be a fasta file containing between 1 and 800,000 sequences (the upper limit is due to memory constraints in the HMMER software).
>54_100 NNNNNNNNNNNNGGGCGATTGNTTTAGCGGCCGCGAATTCGCCCTTTGAAACACCTGTGGTTCTTCCTCCTCCTGGTGGC AGCTCCCAGATGGGTCCTGTCTCAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGGGACCCTGTCCC TCACCTGCGCTGTCTCTGGTGGCTCCATCAGCAGTAGTAACTGGTGGAGTTGGGTCCGCCAGCCCCCAGGGAAGGGGCTG GAGTGGATTGGGGAAATCTATCATAGTGGGAGCACCAACTACAACCCGTCCCTCAAGAGTCGAGTCACCATATCAGTAGA CAAGTCCAAGAACCAGTTCTCCCTGAAGCTGAGCTCTGTGACCGCCGCGGACACGGCCGTGTATTACTGTGCGAGAAAGC TGGGGATTAAGTATGCTTTTGATATCTGGGGCCAAGGGACAATGGTCACCGTCTCTTCAGAGAGTCAGTCCTCCCCAACT GTCTTCCCCCTCGTCTCCTGCGAGAGCCCCCTGTCTGATGAGAATTTGGTGGCCATGGGCTGCCTGGCCCGGGACTTCCT GCCCAGCTCCATTTCCTTCTCCTGGAACTACCAGAACAACACTGAAGTCATGCAGGGTGTCAGAACCTTCCCAACACTGA GGACAGGGGACAAATACACAGCTACCTCGCAGGTGTTACTGTCCGCCAAAAATGTCCTTGAAGGTTCAGATGAATACTTG GTATGCAAAATCCACCATGGCAACAAAAATAAAGATCTGCATGTGCCGATTCCAGCTGTCGTTGAGATGAACCCCAATGT GAGTGTGTTCATTCCACCACGTGATGCCTTCTCTGGCCCTGCACCCCGCAAGTCCAGACTCATCTGCGAGGCCACCAACT TCAGTCCCAAACAGATCACAGTATCCTGGCTACAGGATGGGAAGCCTGTGAAATCTGGCTTCACNNCAGAGCCAGTGACT GTCGANGCCNAANGNATCCAGACCCCAAANCTANNNGTCATNANCNNNCTGACCATCACTGAAAGNAGGGCNANTCNTTT NANCNGCNGGACTAGTCCTTTANTGAGGGNNNNTGAGCTGNCGTANCATGNCATAGCTNNTCNGGNNNGNANTNNNNNTC CNNNTCNNNANNNNNNANNNNNNNNNNNNNNNNNNNNNNNNCNGGGGNNCTANNNNNGNNCTNNNNNNNNNNNNNNNNNN NNNNNNNNNNNNNNNNANNNTCNNNNNNNNNNNNN >70_100 NNNNNNNNNNANNGGGCGATTGATTTAGCGGCCGCGAATTCGCCCTTTCCACGCTCCTGCTGCTGACCATCCCTTCATGG GTCTTGTCCCAGATCACCTTGAAGGAGTCTGGTCCTACGCTGGTGAAACCCACACAGACCCTCACGCTGACCTGCACCTT CTCTGGGTTCTCACTCAGCACTAGTGGAGTGGGTGTGGGCTGGATCCGTCAGCCCCCAGGAAAGGCCCTGGAGTGGCTTG CACTCATTTATTGGAATGATGATAAGCGCTACAGCCCATCTCTGAAGAGCAGGCTCACCATCACCAAGGACACCTCCGAA AACCAGGTGGTCCTTACAATGACCAACATGGACCCTGTGGACACAGCCACATATTACTGTGCACACGGATACAGCTATGG TTACTTCCAGCACTGGGGCCAGGGCACCCTGGTCACCGTCTCCTCAGAGAGTCAGTCCTCCCCAACTGTCTTCCCCCTCG TCTCCTGCGAGAGCCCCCTGTCTGATGAGAATTTGGTGGCCATGGGCTGCCTGGCCCGGGACTTCCTGCCCAGCTCCATT TCCTTCTCCTGGAACTACCAGAACAACACTGAAGTCATGCAGGGTGTCAGAACCTTCCCAACACTGAGGACAGGGGACAA ATACACAGCTACCTCGCAGGTGTTACTGTCCGCCAAAAATGTCCTTGAAGGTTCAGATGAATACTTGGTATGCAAAATCC ACCATGGCAACAAAAACAAAGATCTGCATGTGCCGATTCCAGCTGTCGTTGAGATGAACCCCAATGTGAGTGTGTTCATT CCACCACGTGATGCCTTCTCTGGCCCTGCACCCCGCAAGTCCAGACTCATCTGCGAGGCCACCAACTTCAGTCCCAAACA GATCACAGTATCCTGGCTACAGGATGGGAAGCCTGTGAAATCTGGCTTCACCACAGANCCAGTGACTGTCGANGCCAAAG GATCCAGACCCCNAACCTACNNGGTCATAAGCACACTGACCATCACTGAAAGCANGGCNANNCGTTNNAANNTGCAGGAC TAGTCCCTTTNNTGAGGTNATNNNNANCTNNNTANCATGNCATAGCTGTTNCTGGNNNGAAATGTNNCNGCTNNNNNNCN NNNANNNNNNNNNNNNNNNNNNNNAAANNCNGGGNNGNCNANGNNNNNNTAANNCNNNNANNNGNNNNNNNNNNNNNNNN NCNNNNNGGNNNNNNNNGCNNNNNNNCNNTAANGNANNNGGNNNNN
Output is a fasta file containing translated variable domains and headers describing the segment and CDR composition.
>54_100_frame_0;IGHV4-4 294 0;;IGHJ3 48 0;CARKLGIKYAFDIW;;IGHM 22 1 QVQLQESGPGLVKPSGTLSLTCAVSGGSISS.sNWWSWVRQPPGKGLEWIGEIYHSGSTNYNPSLKSRVTISVDKSKNQFSLKLSSVTAADTAVYYCARKLgi.kyaFDIWGQGTMVTVSS >70_100_frame_2;IGHV2-5 297 1;IGHD5-5/5-18 18 0;IGHJ1 46 0;CAHGYSYGYFQHW;;IGHM 22 1 QITLKESGPTLVKPTQTLTLTCTFSGFSLSTsgVGVGWIRQPPGKALEWLALIYWNDDKRYSPSLKSRLTITKDTSENQVVLTMTNMDPVDTATYYCAHGYsy..gyFQHWGQGTLVTVSS
Development
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