Manual Reference Pages – HLAminer - Derivation of HLA (Human Leukocyte Antigen) class I and class II predictions from shotgun sequence datasets
- This manual assumes that you have a working knowledge of unix, and some shell and perl scripting experience
HLAminer - Derivation of HLA class I and class II predictions from shotgun sequence datasets
- SYNOPSIS
- LICENSE
- OVERVIEW
- DESCRIPTION
- INSTALL
- COMMANDS AND OPTIONS
- PREDICTING FROM LONG (NANOPORE/PACBIO) READS
- DATABASES
- AUTHORS
- CITING
- FULL LICENSE
HLAminer is a pipeline for predicting Human Leukocyte Antigen (HLA) signatures from shotgun sequence data (ie. whole genome, whole transcriptome/RNA-Seq, exome), at the group and allele resolution.
It supports predictions from a variety of DNA sequencing technologies including those from Illumina, MGI, PacBio and Oxford Nanopore.
Predictions are either derived from targeted assembly or direct alignment.
For quick tests on Illumina RNA-seq data:
- Copy ./test-demo/ eg. cp -rf test-demo foo
- In folder "foo", edit the patient.fof file to point to your NGS RNAseq data. Ensure all paths are ok.
- For HLA Predictions by Targeted Assembly of Shotgun Reads: execute ./HLAminer/foo/HPTASRrnaseq.sh For HLA Predictions by Read Alignment: execute ./HLAminer/foo/HPRArnaseq.sh
HLAminer Copyright (c) 2011-present Canada's Michael Smith Genome Science Centre. All rights reserved. TASR Copyright (c) 2010-2022 Canada's Michael Smith Genome Science Centre. All rights reserved. SSAKE Copyright (c) 2006-2022 Canada's Michael Smith Genome Science Centre. All rights reserved.
Due to the clinical implications of HLAminer, the code is now released under the BC Cancer Agency software license agreement (academic use). Details of the license can be accessed at: and at the bottom of this readme file
For commercial licensing options, please contact Patrick Rebstein [email protected]
Software components of HLAminer (eg. TASR) are still distributed under the terms of the GNU General Public License
Derivation of HLA class I and class II predictions from shotgun sequence datasets (HLAminer) by:
- Targeted Assembly of Shotgun Reads (HPTASR)
- Read Alignment (HPRA)
BEST SHORT READ RESULTS ARE OBTAINED WITH HPTASR WITH READS 100bp AND UP (IDEALLY 150bp). IT WILL WORK WITH SHORTER READS (50bp) BUT 4-digit HLA ALLELE PREDICTIONS MAY BE AMBIGUOUS
This clip summarizes the pipeline: https://www.youtube.com/watch?v=j-g8Geh5ST8&list=LL&index=110
The HLA prediction by targeted assembly of short sequence reads (HPTASR), performs targeted de novo assembly of HLA NGS reads and align the resulting contigs to reference HLA alleles from the IMGT/HLA sequence repository using commodity hardware with standard specifications (<2GB RAM, 2GHz). Putative HLA types are inferred by mining and scoring the contig alignments and an expect value is determined for each. The method is accurate, simple and fast to execute and, for transcriptome data, requires low depth of coverage. Known HLA class I/class II reference sequences available from the IMGT/HLA public repository are read by TASR using default options (Warren and Holt 2011) to create a hash table of all possible 15 nt words (k-mers) from these reference sequences. Note that this parameter is customizable and larger k values will yield predictions with increased specificity (at the possible expense of sensitivity). Subsequently, NGS data sets are interrogated for the presence of one of these kmers (on either strand) at the 5’ or 3’ start. Whenever an HLA word is identified, the read is recruited as a candidate for de novo assembly. Upon de novo assembly of all recruited reads, a set of contigs is generated. Only sequence contigs equal or larger than 200nt in length are considered for further analysis, as longer contigs better resolve HLA allelic variants. Reciprocal BLASTN alignments are performed between the contigs and all HLA allelic reference sequences. HPTASR mines the alignments, scoring each possible HLA allele identified, computing and reporting an expect value (E-value) based on the chance of contigs characterizing given HLA alleles and, reciprocally, the chance of reference HLA alleles aligning best to certain assembled contig sequences
The HLA prediction from direct read alignment (HPRA) method is conceptually simpler and faster to execute, since paired reads are aligned up-front to reference HLA alleles. Alignments from the HPTASR and HPRA methods are processed by the same software (HLAminer.pl) to derive HLA-I predictions by scoring and evaluating the probability of each candidate bearing alignments.
Ability to stream the (.sam) output of modern read aligners, directly into HLAminer. Initial support for predicting HLA types from long nanopore reads such as those from Oxford Nanopore Technologies. Better information/sub-routine/date tracking in hlaminer
A more concise HLA allele summary in HLAminer_HPTASR.csv and HLAminer_HPRA.csv (associated .log is unchanged and lists all predictions) Keeps top two [highest-scoring by HLA group] predictions per gene and only the 'P' designated allele when the summary include HLA Sequences reported to have the same antigen binding domain. For the original output, refer to the HLAminer_v1-2.pl included in the ./bin directory A prediction example from MCF-7 PacBio RNA-seq reads is also provided
Updated all HLA sequence databases Corrected shell script that download HLA sequences to reflect change of location at EBI (ie. fasta sub folder) Added support for predictions from direct alignment of single-end reads
1. Download and decompress the tar ball gunzip HLAminer_v1-4.tar.gz tar -xvf HLAminer_v1-4.tar 2. Make sure you see the following directories: ./bin ./databases ./docs ./test-demo 3. Read the docs in the ./docs/ folder 4. Change/Add/Adjust the perl shebang line of each .pl and .sh script in the ./bin/ folder as needed
From direct Read Alignment (HPRA, faster but less accurate): HPRArnaseq_classI.sh HPRArnaseq_classI-II.sh HPRAwgs_classI.sh HPRAwgs_classI-II.sh -and for single end reads- HPRArnaseq_classI_SE.sh HPRArnaseq_classI-II_SE.sh HPRAwgs_classI_SE.sh HPRAwgs_classI-II_SE.sh
From Targeted Assembly (HPTASR, longer but more accurate): HPTASRrnaseq_classI.sh HPTASRrnaseq_classI-II.sh HPTASRwgs_classI.sh HPTASRwgs_classI-II.sh
*Running HPTASRwgs(rnaseq)_classI-II.sh will take longer than HPTASRwgs(rnaseq)_classI.sh, due to the reciprocal BLAST step. You may remove this step from the former (and HLAminer.pl command) to speed things up. However, this step is helpful in weeding out spurious alignments to HLA references. That said, if you're solely interested in HLA-I, you have the option to run the latter set of scripts [HPTASRwgs(rnaseq)_classI.sh].
Also, in the ncbiBlastConfig2-2-XX.txt files (bin and test-demo directories), you may adjust the number of threads and number of reported alignments to speed things up. The options have different name depending on the blast version, refer to the blast manual eg. v2.2.22 option:description -a:threads -v:number of descriptions -b:number of alignments
v2.2.28 -num_threads:threads -max_target_seqs:number of hit sequences to report (when output is 5/xml)
In our hands, a few tests show that blast 2.2.22 may be faster than blast+ (2.2.28) while producing accurate results - HLAminer (Warren et al. 2012) was thoroughly tested with 2.2.22
NCBI blast may be downloaded from: ftp://ftp.ncbi.nlm.nih.gov/blast/executables/blast+/ -or- ftp://ftp.ncbi.nlm.nih.gov/blast/executables/release/
HLAminer.pl
parseXMLblast.pl
- You must install perl module Bio::SearchIO to use HPTASR
- Edit the fullpath location of bwa and other software dependencies in the shell scripts in the ./bin/ folder, as needed
- For your convenience, ncbi blastall and formatdb have been placed in the ./bin/ folder and executed from the following shell scripts:
NAME,PROCESS,NGS DATA TYPE,PREDICTIONS HPRArnaseq_classI.sh,Paired read alignment,RNAseq (transcriptome),HLA-I A,B,C genes HPRArnaseq_classI-II.sh,Paired read alignment,RNAseq (transcriptome),HLA-I A,B,C and HLA-II DP,DQ,DR genes
HPRAwgs_classI.sh,Paired read alignment,Exon capture (exome) and WGS (genome),HLA-I A,B,C genes HPRAwgs_classI-II.sh,Paired read alignment,Exon capture (exome) and WGS (genome),HLA-I A,B,C and HLA-II DP,DQ,DR genes
HPTASRrnaseq_classI.sh,Targeted assembly of sequence reads,RNAseq (transcriptome),HLA-I A,B,C genes HPTASRrnaseq_classI-II.sh,Targeted assembly of sequence reads,RNAseq (transcriptome),HLA-I A,B,C and HLA-II DP,DQ,DR genes
HPTASRwgs_classI.sh,Targeted assembly of sequence reads,Exon capture (exome) and WGS (genome),HLA-I A,B,C genes HPTASRwgs_classI-II.sh,Targeted assembly of sequence reads,Exon capture (exome) and WGS (genome),HLA-I A,B,C and HLA-II DP,DQ,DR genes
Run those scripts by specifying the relative path ../bin/blastall or ../bin/formatdb in the shell scripts AND config file "ncbiBlastConfig.txt". Make sure HPRA and HPTASR are running in same-level directories to ../bin/ (eg. ../test-demo/)
- Before running on your data, inspect the ./test-demo/ folder and familiarize yourself with the files and the execution.
- When you are ready and the demo works well, place the fullpath location of your short read fastq or fasta files in the "patient.fof" file.
- Make sure that the following files are in your working directory:
patient.fof ncbiBlastConfig.txt (specific to the version of blast you are using, see in ../bin and ../test-demo directories
sudo perl -MCPAN -e shell install CJFIELDS/BioPerl-1.6.923.tar.gz change shebang line in all PERL (.pl) scripts and location of bioperl on your system in HLAminer.pl
download and install blast-2.2.22-universal-macosx.tar.gz change path to blast in ncbiconfig.txt
ruby -e "$(curl -fsSL https://raw.githubusercontent.com/Homebrew/install/master/install)" Since databases were indexed with older version, had to re-index: bwa index -a is HLA_ABC_CDS.fasta
change path to bwa in HPRA* shell scripts
TEST YOUR INSTALL BY RUNNING THE SCRIPT IN test-demo. CONSULT THE README-FIRST.txt FILE
Usage: ../bin/HLAminer.pl [v1.4] Derivation of HLA class I and II predictions from shotgun sequence datasets -------------------------------------------------------------------- HPTASR (HLA Predictions by Targeted Assembly of Shotgun Reads): -b blastn alignments......................... -r reciprocal blastn......................... -c contig fasta file......................... -z minimum contig size.......................<200> ------------------------------- OR --------------------------------- HPRA (HLA Predictions by Read Alignment): -a sam alignments............................< -a ngs_vs_hla.sam > or < -a stream > -e single-end reads used (1=yes/0=no)........<0> -------------------------------------------------------------------- -h hla fasta file............................ -p P-designation file........................ -i minimum % sequence identity...............<99> -q minimum log10 (phred-like) expect value...<30> -s minimum score.............................<1000> -n consider null alleles (1=yes/0=no)........<0> -l label (run name) -optional- The shell scripts are set to filter out short (<200) contigs that could blur HPTASR predictions. Feel free to adjust as you see fit. Likewise, HLAminer.pl runs with the set defaults: -z minimum contig size.......................<200> (HPTASR) -i minimum % sequence identity...............<99> (HPTASR / HPRA) -q minimum log10 (phred-like) expect value...<30> (HPTASR / HPRA) -s minimum score.............................<1000> (HPTASR / HPRA) -n consider null alleles (1=yes/0=no)........<0> (HPTASR / HPRA)
The minimum sequence identity applies to the short read paired alignment or blast alignment, depending on the choice made. HLA predictions with a phred-like expect value lower than -q or a score lower than -s will not be diplayed. Because IMGT/HLA reports numerous null alleles, an option exist to consider or not these unexpressed alleles.
Likewise, TASR-based (Targeted Assembly of Short Reads) predictions could be improved by using larger k values for assembly (-k). Experimentation for choosing the ideal k to use depends on the input read length and is warranted.
HLAminer v1.4 provides initial support for HLA prediction from raw uncorrected shotgun nanopore long reads (such as those from Oxford Nanopore Technologies). HLAminer v1.4 implements a streaming approach to reading .sam alignment files, supporting the alignment of GB worth of read data in a few hours and predictions within seconds of alignment completion, without saving costly .sam files to disk.
We tested the software on the NA19240 WGS promethion dataset (2018, older chemistry). https://gigabaseorgigabyte.wordpress.com/2018/05/24/promethion-human-genome-na19240/ with data available from ENA at this location: https://www.ebi.ac.uk/ena/data/view/PRJEB26791
- First, we downloaded read files (ERR2585112 and ERR2585115 from the ENA)
nohup wget ftp://ftp.sra.ebi.ac.uk/vol1/fastq/ERR258/005/ERR2585115/ERR2585115.fastq.gz & nohup wget ftp://ftp.sra.ebi.ac.uk/vol1/fastq/ERR258/002/ERR2585112/ERR2585112.fastq.gz &
- Then, we predicted HLA by running minimap2 and HLAminer v1.4:
/usr/bin/time -v -o minimap_hlaminerERR2585115-1mod.time minimap2 -t 60 -ax map-ont --MD ../database/GCA_000001405.15_GRCh38_genomic.chr-only-noChr6-HLA-I_II_GEN.fa.gz ERR2585115.fastq.gz | ./HLAminer.pl -h ../database/HLA-I_II_GEN.fasta -s 500 -q 1 -i 1 -p ../database/hla_nom_p.txt -a stream
A test is provided at ./test-demo/HPRAwgs_ONTclassI-IIdemo.sh
The output files: HLAminer_HPRA_ERR2585115.csv -and- HLAminer_HPRA_ERR2585115.log
you generate should be comparable* to: HLAminer_HPRA_ERR2585115_test.csv -and- HLAminer_HPRA_ERR2585115_test.log
*exact scores are not to be expected, because they vary based on the HLA sequence database used, which differ from different release of the code (and your own updating of HLA sequence databases, which is highly recommended to do before you use HLAminer).
Results below demonstrate HLAminer's ability to fairly accurately predict HLA-I and -II types from direct [and streamed] nanopore sequencing reads [old chemistry] alignments
HLAminer PREDICTIONS (top 2 per HLA allele group, by high-score): -------------------- A*30:106/A*68:02P B*35:01P/B*57:01P ex. B*57:03P C*04:01P/C*18:01P F*01:01P G*01:01P DQA1*01:02P/DQA1*05:01P DQB1*05:02P/DQB1*03:03P ex. DQB1*03:01P DRB1*12:01P/DRB1*16:02P DPA1*02:02P/DPA1*01:03P DPB1*90:01P/DPB1*01:01P DRA*01:01P REPORTED TYPES (extracted from https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5804087/#MOESM1 Supp file 1 13059_2018_1388_MOESM1_ESM.xlsx Tab S6, NA19240 (Child)): -------------- A*30:01:01G/A*68:02:01G B*35:01:01G/B*57:03:01G C*04:01:01G/C*18:01:01G F*01:01:01:08;F*01:01:01:09;F*01:02;F*01:01:01:10;F*01:03:01:01;F*01:01:01:11;F*01:03:01:02;F*01:01:01:12;F*01:01:02:01;F*01:01:02:02;F*01:01:02:03;F*01:01:02:04;F*01:01:02:05;F*01:01:02:06;F*01:01:01:01;F*01:01:01:02;F*01:01:01:03;F*01:01:01:04;F*01:01:01:05;F*01:01:01:06;F*01:01:01:07/F*01:01:01:08;F*01:01:01:09;F*01:02;F*01:01:01:10;F*01:03:01:01;F*01:01:01:11;F*01:03:01:02;F*01:01:01:12;F*01:01:02:01;F*01:01:02:02;F*01:01:02:03;F*01:01:02:04;F*01:01:02:05;F*01:01:02:06;F*01:01:01:01;F*01:01:01:02;F*01:01:01:03;F*01:01:01:04;F*01:01:01:05;F*01:01:01:06;F*01:01:01:07 G*01:06;G*01:01:02:01;G*01:01:02:02;G*01:18;G*01:08:01;G*01:01:18;G*01:01:19/G*01:05N H*02:05/H*02:05 J*02:01;J*01:01:01:05;J*01:01:01:04;J*01:01:01:03;J*01:01:01:02;J*01:01:01:01/J*02:01;J*01:01:01:05;J*01:01:01:04;J*01:01:01:03;J*01:01:01:02;J*01:01:01:01 L*01:01:02;L*01:01:01:01;L*01:01:01:02;L*01:01:01:03/L*01:02 DQA1*01:02:01G/DQA1*05:01:01G DQB1*05:02:01G/DQB1*03:01:01G DRB1*12:01:01G/DRB1*16:02:01G DPA1*02:01:01G/DPA1*02:02:02G DPB1*01:01:01G/DPB1*01:01:01G DOA*01:01:05/DOA*01:01:02:03;DOA*01:01:02:02;DOA*01:01:02:01;DOA*01:01:04:02;DOA*01:01:04:01 DMA*01:01:01:02;DMA*01:01:01:01;DMA*01:04;DMA*01:03;DMA*01:02;DMA*01:01:01:04;DMA*01:01:01:03/DMA*01:01:01:02;DMA*01:01:01:01;DMA*01:04;DMA*01:03;DMA*01:02;DMA*01:01:01:04;DMA*01:01:01:03 DMB*01:02;DMB*01:01:01:04;DMB*01:01:01:03;DMB*01:01:01:02;DMB*01:03:01:04;DMB*01:01:01:01;DMB*01:03:01:03;DMB*01:03:01:02;DMB*01:03:01:01;DMB*01:05;DMB*01:04/DMB*01:02;DMB*01:01:01:04;DMB*01:01:01:03;DMB*01:01:01:02;DMB*01:03:01:04;DMB*01:01:01:01;DMB*01:03:01:03;DMB*01:03:01:02;DMB*01:03:01:01;DMB*01:05;DMB*01:04 DRA*01:01:01:01;DRA*01:02:01;DRA*01:01:01:02;DRA*01:02:02;DRA*01:01:01:03;DRA*01:02:03;DRA*01:01:02/DRA*01:01:01:01;DRA*01:02:01;DRA*01:01:01:02;DRA*01:02:02;DRA*01:01:01:03;DRA*01:02:03;DRA*01:01:02
For more information, please refer to:
Follow these instructions to download updated HLA sequences from ebi/imgt (shell scripts to automatically download and format the databases exist in ./database/) and refer to README.txt in the ./database directory:
- Coding HLA sequences
HLA CDS sequences from:
wget ftp://ftp.ebi.ac.uk/pub/databases/imgt/mhc/hla/fasta/A_nuc.fasta
wget ftp://ftp.ebi.ac.uk/pub/databases/imgt/mhc/hla/fasta/B_nuc.fasta
wget ftp://ftp.ebi.ac.uk/pub/databases/imgt/mhc/hla/fasta/C_nuc.fasta
cat A_nuc.fasta B_nuc.fasta C_nuc.fasta | perl -ne 'chomp;if(/\>\S+\s+(\S+)/){print ">$1\n";}else{print "$_\n";}' > HLA_ABC_CDS.fasta
../bin/formatdb -p F -i HLA_ABC_CDS.fasta
/home/pubseq/BioSw/bwa/bwa-0.5.9/bwa index -a is HLA_ABC_CDS.fasta
- HLA genomic sequences
To make the HLA genomic sequence database, execute these unix commands:
wget ftp://ftp.ebi.ac.uk/pub/databases/imgt/mhc/hla/fasta/A_gen.fasta
wget ftp://ftp.ebi.ac.uk/pub/databases/imgt/mhc/hla/fasta/B_gen.fasta
wget ftp://ftp.ebi.ac.uk/pub/databases/imgt/mhc/hla/fasta/C_gen.fasta
cat A_gen.fasta B_gen.fasta C_gen.fasta | perl -ne 'chomp;if(/\>\S+\s+(\S+)/){print ">$1\n";}else{print "$_\n";}' > HLA_ABC_GEN.fasta
../bin/formatdb -p F -i HLA_ABC_GEN.fasta
/home/pubseq/BioSw/bwa/bwa-0.5.9/bwa index -a is HLA_ABC_GEN.fasta
FOR YOUR CONVENIENCE, A SINGLE SHELL SCRIPT CAN BE RUN FROM ../database TO UPDATE ALL IMGT-HLA SEQUENCE DATABASES AND CREATE BWA/BLAST INDEXES. JUST KEEP IN MIND THAT THIS IS DONE WITH A SPECIFIC VERSION OF THESE TOOLS, IF YOU UPGRADE, YOU WILL HAVE TO REGENERATE THE INDEXES
../database/updateAll.sh
Note: For alignment of nanopore genomic/transcriptomic reads, we recommend aligning to a file comprised of human genome chromosome and HLA-I and -II alleles to reduce the noise in alignments (especially when running minimap2). If you are predicting from direct ONT (nanopore) or PacBio long read alignments, please update the genome files:
cd database cat GCA_000001405.15_GRCh38_genomic.chr-only-noChr6.fa HLA-I_II_GEN.fasta | pigz - > GCA_000001405.15_GRCh38_genomic.chr-only-noChr6-HLA-I_II_GEN.fa.gz cat GCA_000001405.15_GRCh38_genomic.chr-only-noChr6.fa HLA-I_II_CDS.fasta | pigz - > GCA_000001405.15_GRCh38_genomic.chr-only-noChr6-HLA-I_II_CDS.fa.gz
Genome file (without Chr6) https://www.bcgsc.ca/downloads/btl/hlaminer/GCA_000001405.15_GRCh38_genomic.chr-only-noChr6.fa.gz
or download those files (Aug 2022 update) from: https://www.bcgsc.ca/downloads/btl/hlaminer/
- P designation files
Upgrade the P designation info from:
info: http://hla.alleles.org/wmda/index.html
file: http://hla.alleles.org/wmda/hla_nom_p.txt
HLA predictions from read pair alignments:
HLAminer_HPRA.log HLAminer_HPRA.csv
HLA predictions from targeted assemblies:
HLAminer_HPTASR.log HLAminer_HPTASR.csv
The .log file tracks the process of HLA mining. It contains the following information: -HLAminer command and parameters utilized -Contig/read pair alignment output and best HLA hit for each -Initial gene summary, score and expect value -Final summary, listing all predictions by highest score (more likely).
The .csv file contains HLAminer predictions. Predictions are listed by HLA gene and ranked by highest score. Predictions 1) and 2) are expected to represent the two alleles for each.
eg.
----------------------------------------------------------------------
SUMMARY
MOST LIKELY HLA-I ALLELES (Confidence (-10 * log10(Eval)) >= 30, Score >= 500)
Allele,Score,Expect (Eval) value,Confidence (-10 * log10(Eval))
----------------------------------------------------------------------
HLA-A
Prediction #1 - A*26
A*26:33,4179,5.22e-124,1232.8
Prediction #2 - A*33
A*33:24,1791,2.41e-75,746.2
Prediction #3 - A*68
A*68:05,597,1.85e-10,97.3
From these predictions, the individual is expected to be heterozygous with HLA-I A alleles A26 and A33. The chance, expect value and confidence are different representations of the same metric. The Confidence represents the Expect value - Eval - as a score in a manner analoguous to the phred score employed in sequencing to quickly assess the likelihood of a base being correct.
Predictions/read pair are ambiguous when there are multiple predicted allele groups and/or protein coding alleles with the same score.
Rene Warren
Thank you for your 
If you use HLAminer for you research, please cite:
Warren RL, Choe G, Freeman DJ, Castellarin M, Munro S, Moore R, Holt RA. 2012. Derivation of HLA types from shotgun sequence datasets. Genome Med. 4:95
and
Warren RL. 2022. HLA predictions from long sequence read alignments, streamed directly into HLAminer. arXiv. https://doi.org/10.48550/arXiv.2209.09155
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INTELLECTUAL PROPERTY RIGHTS: Subject to Section 5 below, all patents, copyrights, trade secrets, service marks, trademarks and other proprietary rights in or related to the Product and any improvements, modifications and enhancements thereof are and will remain the exclusive property of BCCA or its licensors. You agree that You will not, either during or after the termination of this Agreement, contest or challenge the title to or the intellectual property rights of BCCA or its licensors in the Product or any portion thereof.
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OWNERSHIP OF IMPROVEMENTS: In the event that the Product, in the form provided to You, includes source code (the "Source Code"), You are entitled to make improvements, modifications and enhancements to the Source Code (collectively, "Improvements") which Improvements are to be used by You for non-profit research and educational purposes only and You shall be the owner of those Improvements that You directly make and of all intellectual property rights to such Improvements, subject to the foregoing limits on Your use and distribution of such Improvements. You hereby grant to BCCA a perpetual, non-exclusive, worldwide, fully-paid, irrevocable license to use such Improvements for any purposes whatsoever, and to sublicense such Improvements including the right for third parties to sublicense the same, in perpetuity to the extent such rights are not limited in duration under applicable law, without identifying or seeking Your consent. Notwithstanding the foregoing, You acknowledge that BCCA and its licensors will retain or own all rights in and to any pre-existing code or other technology, content and data that may be incorporated in the Improvements. For greater certainty, this Section applies solely to the Source Code and shall not give You any rights with respect to the object code or any other portion or format of the Product which use, for greater certainty, is limited as set forth in this Agreement including as set out in Section 3(b) above. You acknowledge and agree that you will provide copies of Improvements to BCCA in such format as reasonably requested by BCCA at any time upon the request of BCCA.
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CONFIDENTIALITY: You acknowledge that the Product is and incorporates confidential and proprietary information developed, acquired by or licensed to BCCA. You will take all reasonable precautions necessary to safeguard the confidentiality of the Product, and will not disclose any information about the Product to any other person without BCCA's prior written consent. You will not allow the removal or defacement of any confidential or proprietary notice placed on the Product. You acknowledge that any breach of this Section 6 will cause irreparable harm to BCCA and its licensors.
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NO WARRANTIES: THIS PRODUCT IS PROVIDED TO YOU BY BCCA IN ORDER TO ALLOW YOU TO OBTAIN ACCESS TO LEADING ACADEMIC RESEARCH. THE PRODUCT IS PROVIDED TO YOU ON AN "AS IS" BASIS WITHOUT WARRANTY OF ANY KIND. NO WARRANTY, REPRESENTATION OR CONDITION EITHER EXPRESS OR IMPLIED, INCLUDING WITHOUT LIMITATION, ANY IMPLIED WARRANTY OR CONDITION OF MERCHANTABILITY, NON-INFRINGEMENT, PERFORMANCE, DURABILITY OR FITNESS FOR A PARTICULAR PURPOSE OR USE SHALL APPLY. BCCA DOES NOT WARRANT THAT THE PRODUCT WILL OPERATE ON A CONTINUOUS OR TROUBLE FREE BASIS.
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LIMITATION OF LIABILITY: TO THE MAXIMUM EXTENT PERMITTED BY APPLICABLE LAW, IN NO EVENT SHALL THE AGGREGATE LIABILITY OF BCCA TO YOU EXCEED THE AMOUNT YOU HAVE PAID TO ACQUIRE THE PRODUCT ("MAXIMUM AMOUNT") AND WHERE YOU HAVE NOT PAID ANY AMOUNT FOR THE PRODUCT THEN THE MAXIMUM AMOUNT SHALL BE DEEMED TO BE CDN$100.00. IN NO EVENT SHALL BCCA BE LIABLE FOR ANY INDIRECT, INCIDENTAL, CONSEQUENTIAL, OR SPECIAL DAMAGES, INCLUDING WITHOUT LIMITATION ANY DAMAGES FOR LOST PROFITS OR SAVINGS, REGARDLESS OF WHETHER THEY HAVE BEEN ADVISED OF THE POSSIBILITY OF SUCH DAMAGE. EXCEPT TO THE EXTENT THAT THE LAWS OF A COMPETENT JURISDICTION REQUIRE LIABILITIES BEYOND AND DESPITE THESE LIMITATIONS, EXCLUSIONS AND DISCLAIMERS, THESE LIMITATIONS, EXCLUSIONS AND DISCLAIMERS SHALL APPLY WHETHER AN ACTION, CLAIM OR DEMAND ARISES FROM A BREACH OF WARRANTY OR CONDITION, BREACH OF CONTRACT, NEGLIGENCE, STRICT LIABILITY OR ANY OTHER KIND OF CIVIL OR STATUTORY LIABILITY CONNECTED WITH OR ARISING FROM THIS AGREEMENT. YOU AGREE THAT THE FOREGOING DISCLAIMER OF WARRANTIES AND LIMITATION OF LIABILITY ARE FAIR IN LIGHT OF THE NATURE OF THE RIGHTS GRANTED HEREIN AND THE AMOUNT OF FEES PAID BY YOU IN RESPECT OF THE PRODUCT.
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INDEMNITY: You will indemnify, defend and hold harmless BCCA, its board of directors, staff and agents from and against any and all liability, loss, damage, action, claim or expense (including attorney's fees and costs at trial and appellate levels) in connection with any claim, suit, action, demand or judgement (collectively, "Claim") arising out of, connected with, resulting from, or sustained as a result of Your use of the Product or the downloading of the Product, including without limitation, any Claim relating to infringement of BCCA's intellectual property rights or the intellectual property rights of any third party.
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SUPPORT AND MAINTENANCE: You acknowledge and agree that, unless and to the extent expressly agreed by BCCA in a separate written document, the Product is provided to You without any support or maintenance from BCCA and, for greater certainty, BCCA shall have no obligation to issue any update or upgrade to any Product.
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TERM: This Agreement is effective until terminated. You may terminate this Agreement at any time by ceasing use of the Product and destroying or deleting any copies of the Product. This Agreement will terminate immediately without notice from BCCA if You fail to comply with any provision of this Agreement. BCCA may terminate this Agreement at any time upon notice to you where BCCA determines, in its sole discretion, that any continued use of the Product could infringe the rights of any third parties. Upon termination of this Agreement, and in any event upon BCCA delivering You notice of termination, You shall immediately purge all Products from Your computer system(s), return to BCCA all copies of the Product that are in Your possession or control, and cease any further development of any Improvements. On any termination of this Agreement Sections 1, 4, 6, 7, 8, 9, 13 and 14 shall survive such termination.
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GOVERNMENT END USERS: Where any of the Product is used, duplicated or disclosed by or to the United States government or a government contractor or sub contractor, it is provided with RESTRICTED RIGHTS as defined in Title 48 CFR 52.227-19 and is subject to the following: Title 48 CFR 2.101, 52.227-19, 227.7201 through 227.7202-4, FAR 52.227-14, and FAR 52.227-19(c)(1-2) and (6/87), and where applicable, the customary software license, as described in Title 48 CFR 227-7202 with respect to commercial software and commercial software documentation including DFAR 252.227-7013, DFAR 252,227-7014, DFAR 252.227-7015 and DFAR 252.7018, all as applicable.
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USE OF THE DOWNLOAD SERVICE: You acknowledge and agree that you will be responsible for all costs, charges and taxes (where applicable) arising out of Your use of the Product and the downloading of the Product. You acknowledge that You are responsible for supplying any hardware or software necessary to use the Product pursuant to this Agreement.
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GENERAL PROVISIONS: (a) This Agreement will be governed by the laws of the Province of British Columbia, and the laws of Canada applicable therein, excluding any rules of private international law that lead to the application of the laws of any other jurisdiction. The United Nations Convention on Contracts for the International Sale of Goods (1980) does not apply to this Agreement. The courts of the Province of British Columbia shall have non-exclusive jurisdiction to hear any matter arising in connection with this Agreement. (b) USE OF THE PRODUCT IS PROHIBITED IN ANY JURISDICTION WHICH DOES NOT GIVE EFFECT TO THE TERMS OF THIS AGREEMENT. (c) You agree that no joint venture, partnership, employment, consulting or agency relationship exists between You and BCCA as a result of this Agreement or Your use of the Product. (d) You hereby consent to Your contact information and any other personally identifiable information that You provide to us being disclosed to and maintained and used by us and our business partners for the purposes of (i) managing and developing our respective businesses and operations; (ii) marketing products and services to You and your staff; and (iii) developing new and enhancing existing products. You further agree that we may provide this information to other persons as required to satisfy any legal requirements and to any person that acquires some or all of the assets of BCCA. Where any of the personally identifiable information that You provide to us is in respect of individuals other than Yourself (such as Your staff) then You represent and warrant to use that You have obtained all necessary consents and authorizations from such individuals in order to comply with this provision. Please see the BCCA website for further information regarding personally identifiable information. (e) This Agreement is the entire Agreement between You and BCCA relating to this subject matter. You will not contest the validity of this Agreement merely because it is in electronic form. No modification of this Agreement will be binding, unless in writing and accepted by an authorized representative of each party. (f) The provisions of this Agreement are severable in that if any provision in the Agreement is determined to be invalid or unenforceable under any controlling body of law, that will not affect the validity or enforceability of the remaining provisions of the Agreement. (g) You agree to print out or download a copy of this Agreement and retain it for Your records. (h) You consent to the use of the English language in this Agreement. (i) You may not assign this Agreement or any of Your rights or obligations hereunder without BCCA's prior written consent. BCCA, at its sole discretion may assign this Agreement without notice to You.