<html><head><meta http-equiv="Content-Type" content="text/html charset=utf-8"></head><body style="word-wrap: break-word; -webkit-nbsp-mode: space; -webkit-line-break: after-white-space;" class=""><div class=""><div class=""></div></div><div class=""><div class=""><br class=""><div><blockquote type="cite" class=""><div class="">26. feb. 2016 kl. 12.41 skrev Anja Thormann <<a href="mailto:anja@ebi.ac.uk" class="">anja@ebi.ac.uk</a>>:</div><br class="Apple-interchange-newline"><div class="">
<meta http-equiv="Content-Type" content="text/html; charset=utf-8" class=""><div style="word-wrap: break-word; -webkit-nbsp-mode: space; -webkit-line-break: after-white-space;" class=""><div class="">Hi Johanne,</div><div class=""><br class=""></div><div class=""><div class="">Thank you very much for the detailed bug report. I could reproduce the problem and we pushed another bug fix to the release/83 branch dealing with the number of open connections. Please update ensembl-variation and ensembl-io repos both for release/83. </div><div class=""><br class=""></div><div class="">The files you see in your working directory are binary index files that are created locally for remote VCF files that store the genotypes. The index files speed up location based lookups and you can delete them after your computations.</div><div class=""><br class=""></div><div class="">We don't support computation of LD values bewteen SNPs located on different chromosomes. The assumption is that different chromosomes segregate independently during meiosis and only genes/alleles located on the same chromosome can be linked.</div></div><div class=""><br class=""></div><div class="">HTH,</div><div class="">Anja</div><br class=""><div class=""><blockquote type="cite" class=""><div class="">On 25 Feb 2016, at 18:00, Johanne Håøy Horn <<a href="mailto:johannhh@ifi.uio.no" class="">johannhh@ifi.uio.no</a>> wrote:</div><br class="Apple-interchange-newline"><div class="">
<div style="word-wrap: break-word; -webkit-nbsp-mode: space; -webkit-line-break: after-white-space;" class="">
Hello again!
<div class=""><br class="">
</div>
<div class="">Thank you for the bug fix. My apologies, I read this old correspondence and mistakenly thought you still calculated LD on the fly in stead of having it in the database: <a href="https://www.biostars.org/p/109785/#147784" class="">https://www.biostars.org/p/109785/#147784</a></div>
<div class="">
<div class=""><br class="">
</div>
<div class="">I use all LD populations for now, as you suggested. However, a couple more questions appeared:</div>
<div class=""><br class="">
</div>
<div class="">1) When I call my $ldfc = $ldfc_adaptor->fetch_by_VariationFeature($vf, $ld_population);, lots of vcf files are downloaded. The terminal prints out «[get_local_version] downloading the index file…» when the files are downloaded, and the following
files appear in my folder:</div>
<div class=""><br class="">
</div>
<div class="">
<div class="">ALL.chr1.phase3_shapeit2_mvncall_integrated_v3plus_nounphased.rsID.genotypes.GRCh38_dbSNP.vcf.gz.tbi</div>
<div class="">ALL.chr10.phase3_shapeit2_mvncall_integrated_v3plus_nounphased.rsID.genotypes.GRCh38_dbSNP.vcf.gz.tbi</div>
<div class="">ALL.chr11.phase3_shapeit2_mvncall_integrated_v3plus_nounphased.rsID.genotypes.GRCh38_dbSNP.vcf.gz.tbi</div>
<div class="">ALL.chr12.phase3_shapeit2_mvncall_integrated_v3plus_nounphased.rsID.genotypes.GRCh38_dbSNP.vcf.gz.tbi</div>
<div class="">ALL.chr14.phase3_shapeit2_mvncall_integrated_v3plus_nounphased.rsID.genotypes.GRCh38_dbSNP.vcf.gz.tbi</div>
<div class="">ALL.chr15.phase3_shapeit2_mvncall_integrated_v3plus_nounphased.rsID.genotypes.GRCh38_dbSNP.vcf.gz.tbi</div>
<div class="">ALL.chr16.phase3_shapeit2_mvncall_integrated_v3plus_nounphased.rsID.genotypes.GRCh38_dbSNP.vcf.gz.tbi</div>
<div class="">ALL.chr19.phase3_shapeit2_mvncall_integrated_v3plus_nounphased.rsID.genotypes.GRCh38_dbSNP.vcf.gz.tbi</div>
<div class="">ALL.chr2.phase3_shapeit2_mvncall_integrated_v3plus_nounphased.rsID.genotypes.GRCh38_dbSNP.vcf.gz.tbi</div>
<div class="">ALL.chr3.phase3_shapeit2_mvncall_integrated_v3plus_nounphased.rsID.genotypes.GRCh38_dbSNP.vcf.gz.tbi</div>
<div class="">ALL.chr4.phase3_shapeit2_mvncall_integrated_v3plus_nounphased.rsID.genotypes.GRCh38_dbSNP.vcf.gz.tbi</div>
<div class="">ALL.chr5.phase3_shapeit2_mvncall_integrated_v3plus_nounphased.rsID.genotypes.GRCh38_dbSNP.vcf.gz.tbi</div>
<div class="">ALL.chr8.phase3_shapeit2_mvncall_integrated_v3plus_nounphased.rsID.genotypes.GRCh38_dbSNP.vcf.gz.tbi</div>
<div class="">ALL.chr9.phase3_shapeit2_mvncall_integrated_v3plus_nounphased.rsID.genotypes.GRCh38_dbSNP.vcf.gz.tbi</div>
</div>
<div class=""><br class="">
</div>
<div class="">What are these files? Why are they downloaded? Will release 84 have a different way of doing it?</div>
<div class=""><br class="">
</div>
<div class="">2) When I try to use a large file of SNPs (example attached in this email), I get the following message after all the 14 vcf-files posted above have been downloaded:</div>
<div class="">
<div class=""><font face="Courier New" class="">[kftp_connect_file]</font></div>
<div class=""><font face="Courier New" class="">[main] fail to open the data file.</font></div>
<div class=""><font face="Courier New" class="">[get_local_version] downloading the index file...</font></div>
<div class=""><font face="Courier New" class="">[kftp_connect_file] 530 You may not have more than 15 concurrent connections at any time.</font></div>
<div class=""><font face="Courier New" class="">[download_from_remote] fail to open remote file.</font></div>
</div>
<div class=""><font face="Courier New" class=""><br class="">
</font></div>
<div class="">If it tries to download another file at the same time the restriction threshold is met, I get this error message:</div>
<div class=""><font face="Courier New" class="">
<div class="">[get_local_version] downloading the index file...</div>
<div class="">[kftp_connect_file] 530 You may not have more than 15 concurrent connections at any time.</div>
<div class="">[download_from_remote] fail to open remote file.</div>
<div class="">[tabix] failed to load the index file.</div>
<div class="">[get_local_version] downloading the index file...</div>
<div class="">Can't use an undefined value as an ARRAY reference at /Users/Johanne/src/ensembl-io/modules/Bio/EnsEMBL/IO/Parser/BaseVCF4.pm line 570, <> line 22.</div>
</font></div>
<div class=""><font face="Courier New" class=""><br class="">
</font></div>
<div class="">Does this mean I cannot connect to all chromosomes at the same time? Does LD only get computed between variants on the same chromosome? What is the reason behind this restriction? I am not too familiar with reasoning behind choices of structure
etc in LD data sets, so these questions are based on my impression that variants can be in LD even though they are located on different chromosomes. If you have a different opinion on this, I would be happy to hear it!</div>
<div class=""><br class="">
</div>
<div class="">My script is the following (basically a copy of your previous suggestions :) )</div>
<div class="">
<div class=""><font face="Courier New" class="">use strict;</font></div>
<div class=""><font face="Courier New" class="">use warnings;</font></div>
<div class=""><font face="Courier New" class="">use Bio::EnsEMBL::Registry;</font></div>
<div class=""><font face="Courier New" class=""><br class="">
</font></div>
<div class=""><font face="Courier New" class="">my $start_run = time();</font></div>
<div class=""><font face="Courier New" class="">open (OUT, ">expandedSNPS.txt");</font></div>
<div class=""><font face="Courier New" class=""><br class="">
</font></div>
<div class=""><font face="Courier New" class="">my $registry = 'Bio::EnsEMBL::Registry';</font></div>
<div class=""><font face="Courier New" class=""><br class="">
</font></div>
<div class=""><font face="Courier New" class="">$registry->load_registry_from_db(</font></div>
<div class=""><font face="Courier New" class=""><span class="Apple-tab-span" style="white-space:pre"></span>-host => '<a href="http://ensembldb.ensembl.org/" class="">ensembldb.ensembl.org</a>',</font></div>
<div class=""><font face="Courier New" class=""><span class="Apple-tab-span" style="white-space:pre"></span>-user => 'anonymous'</font></div>
<div class=""><font face="Courier New" class=""><span class="Apple-tab-span" style="white-space:pre"></span>);</font></div>
<div class=""><font face="Courier New" class=""><br class="">
</font></div>
<div class=""><font face="Courier New" class=""># Connect to the databases:</font></div>
<div class=""><font face="Courier New" class="">my $variation_adaptor = $registry->get_adaptor('homo_sapiens', 'variation', 'variation');</font></div>
<div class=""><font face="Courier New" class="">my $ldfc_adaptor = $registry->get_adaptor('homo_sapiens', 'variation', 'ldfeaturecontainer');</font></div>
<div class=""><font face="Courier New" class="">my $pop_adaptor = $registry->get_adaptor('homo_sapiens', 'variation', 'population');</font></div>
<div class=""><font face="Courier New" class="">$variation_adaptor->db->use_vcf(1);</font></div>
<div class=""><font face="Courier New" class=""><br class="">
</font></div>
<div class=""><font face="Courier New" class="">my $ld_population = $pop_adaptor->fetch_by_name('1000GENOMES:phase_3:CEU');</font></div>
<div class=""><font face="Courier New" class=""><br class="">
</font></div>
<div class=""><font face="Courier New" class=""># Loop through all SNPs available and find SNPs in LD</font></div>
<div class=""><font face="Courier New" class="">while(<>) {</font></div>
<div class=""><span style="font-family: 'Courier New';" class=""><span class="Apple-tab-span" style="white-space:pre"></span>chomp;</span></div>
<div class=""><span class="Apple-tab-span" style="font-family: 'Courier New'; white-space: pre;"></span><span style="font-family: 'Courier New';" class="">my $variation_name = $_;</span></div>
<div class=""><font face="Courier New" class=""><span class="Apple-tab-span" style="white-space:pre"></span>my $variation = $variation_adaptor->fetch_by_name($variation_name);</font></div>
<div class=""><font face="Courier New" class=""><span class="Apple-tab-span" style="white-space:pre"></span>my @var_features = @{ $variation->get_all_VariationFeatures() };</font></div>
<div class=""><font face="Courier New" class=""><br class="">
</font></div>
<div class=""><font face="Courier New" class=""><span class="Apple-tab-span" style="white-space:pre"></span>foreach my $vf (@var_features) {</font></div>
<div class=""><font face="Courier New" class=""><span class="Apple-tab-span" style="white-space:pre"></span>my $rsid = $vf->name;</font></div>
<div class=""><font face="Courier New" class=""><span class="Apple-tab-span" style="white-space:pre"></span>my $start = $vf->start;</font></div>
<div class=""><font face="Courier New" class=""><span class="Apple-tab-span" style="white-space:pre"></span>my $region = $vf->seq_region_name;</font></div>
<div class=""><font face="Courier New" class=""><span class="Apple-tab-span" style="white-space:pre"></span>print OUT "$rsid\t$start\t$region\n";</font></div>
<div class=""><font face="Courier New" class=""><span class="Apple-tab-span" style="white-space:pre"></span>print $ld_population->name, "\n";</font></div>
<div class=""><font face="Courier New" class=""><br class="">
</font></div>
<div class=""><font face="Courier New" class=""><span class="Apple-tab-span" style="white-space:pre"></span>my $ldfc = $ldfc_adaptor->fetch_by_VariationFeature($vf, $ld_population);</font></div>
<div class=""><font face="Courier New" class=""><span class="Apple-tab-span" style="white-space:pre"></span>my @ld_values = @{ $ldfc->get_all_ld_values() };</font></div>
<div class=""><font face="Courier New" class=""><br class="">
</font></div>
<div class=""><font face="Courier New" class=""> <span class="Apple-tab-span" style="white-space:pre">
</span>foreach my $ld_hash (@ld_values) {</font></div>
<div class=""><font face="Courier New" class=""><span class="Apple-tab-span" style="white-space:pre"></span>
<span class="Apple-tab-span" style="white-space:pre"></span>my $r2 = $ld_hash->{r2};</font></div>
<div class=""><font face="Courier New" class=""> <span class="Apple-tab-span" style="white-space:pre">
</span><span class="Apple-tab-span" style="white-space:pre"></span>my $variation_name1 = $ld_hash->{variation1}->variation_name;</font></div>
<div class=""><font face="Courier New" class=""> <span class="Apple-tab-span" style="white-space:pre">
</span><span class="Apple-tab-span" style="white-space:pre"></span>my $variation_name2 = $ld_hash->{variation2}->variation_name;</font></div>
<div class=""><font face="Courier New" class=""> <span class="Apple-tab-span" style="white-space:pre">
</span><span class="Apple-tab-span" style="white-space:pre"></span>print OUT "\t$variation_name1\t$variation_name2\tr2=$r2\n";</font></div>
<div class=""><font face="Courier New" class=""> <span class="Apple-tab-span" style="white-space:pre">
</span>}</font></div>
<div class=""><font face="Courier New" class=""> <span class="Apple-tab-span" style="white-space:pre">
</span>}</font></div>
<div class=""><font face="Courier New" class="">}</font></div>
<div class=""><font face="Courier New" class=""><br class="">
</font></div>
<div class=""><font face="Courier New" class="">close OUT;</font></div>
<div class=""><font face="Courier New" class="">my $end_run = time();</font></div>
<div class=""><font face="Courier New" class="">my $run_time = $end_run - $start_run;</font></div>
<div class=""><font face="Courier New" class="">print "Job took $run_time seconds\n";</font></div>
</div>
<div class=""><br class="">
</div>
<div class=""></div>
</div>
</div>
<span id="cid:7EE5CD546CD6334990ED47FCE1A71CD4@mail.uio.no" class=""><longLD.txt></span><div style="word-wrap: break-word; -webkit-nbsp-mode: space; -webkit-line-break: after-white-space;" class=""><div class=""><div class=""></div><div class=""><br class=""><div class=""><blockquote type="cite" class=""><div class="">25. feb. 2016 kl. 14.24 skrev Anja Thormann <<a href="mailto:anja@ebi.ac.uk" class="">anja@ebi.ac.uk</a>>:</div><br class="Apple-interchange-newline"><div class="">
<div style="word-wrap: break-word; -webkit-nbsp-mode: space; -webkit-line-break: after-white-space;" class="">Hi Johanne,<div class=""><br class=""></div><div class="">thank you for reporting this. The problem was caused by an error in the sql statement. I fixed the wrong statement and pushed the fix again to the release/83 branch. Please pull the changes. However, the call is not going to return any data at the moment. We recently switched from using a database for storing genotypes to reading the the genotypes from VCF files instead. This functionality is currently not supported by the method ($vf->get_all_LD_Populations()). We will add this for release/84. In the meantime please use the LDFeatureContainerAdaptor and loop over all the populations for which we could compute LD data. The populations can be retrieved from the population adaptor using fetch_all_LD_Populations. We compute LD data on 1000 Genomes phase 3 data. In addition to computing LD data for a given SNP you can also specify a region or a a set of SNPs for which you want to compute LD data. Use the following methods from the LDFeatureContainerAdaptor (<a href="http://www.ensembl.org/info/docs/Doxygen/variation-api/classBio_1_1EnsEMBL_1_1Variation_1_1DBSQL_1_1LDFeatureContainerAdaptor.html" class="">http://www.ensembl.org/info/docs/Doxygen/variation-api/classBio_1_1EnsEMBL_1_1Variation_1_1DBSQL_1_1LDFeatureContainerAdaptor.html</a>): fetch_by_VariationFeatures, fetch_by_Slice.</div></div></div></blockquote><blockquote type="cite" class=""><div style="word-wrap: break-word; -webkit-nbsp-mode: space; -webkit-line-break: after-white-space;" class=""><div class=""><br class=""></div><div class="">Best,</div><div class="">Anja</div><div class=""><br class=""><div class=""><blockquote type="cite" class=""><div class="">On 25 Feb 2016, at 09:57, Johanne Håøy Horn <<a href="mailto:johannhh@ifi.uio.no" class="">johannhh@ifi.uio.no</a>> wrote:</div><br class="Apple-interchange-newline"><div class="">
<div style="word-wrap: break-word; -webkit-nbsp-mode: space; -webkit-line-break: after-white-space;" class="">
<div class="">Dear Ensembl team, </div>
<div class=""><br class="">
</div>
<div class="">When I do the following call: my @vf_pops = @{ $vf->get_all_LD_Populations() }; I get this error:</div>
<div class=""><font face="Courier New" class="">DBD::mysql::st execute failed: Unknown column 'ip.population_id' in 'field list' at /Users/Johanne/src/ensembl-variation/modules/Bio/EnsEMBL/Variation/VariationFeature.pm line 1429, <> line 1.DBD::mysql::st execute
failed: Unknown column 'ip.population_id' in 'field list' at /Users/Johanne/src/ensembl-variation/modules/Bio/EnsEMBL/Variation/VariationFeature.pm line 1429, <> line 1.</font></div>
<div class=""><br class="">
</div>
<div class="">Here’s the full script:</div>
<div class="">
<div class=""><font face="Courier New" class="">use strict;</font></div>
<div class=""><font face="Courier New" class="">use warnings;</font></div>
<div class=""><font face="Courier New" class="">use Bio::EnsEMBL::Registry;</font></div>
<div class=""><font face="Courier New" class=""><br class="">
</font></div>
<div class=""><font face="Courier New" class="">my $start_run = time();</font></div>
<div class=""><font face="Courier New" class=""><br class="">
</font></div>
<div class=""><font face="Courier New" class="">my $registry = 'Bio::EnsEMBL::Registry';</font></div>
<div class=""><font face="Courier New" class=""><br class="">
</font></div>
<div class=""><font face="Courier New" class="">$registry->load_registry_from_db(</font></div>
<div class=""><font face="Courier New" class=""> -host => '<a href="http://ensembldb.ensembl.org/" class="">ensembldb.ensembl.org</a>',</font></div>
<div class=""><font face="Courier New" class=""> -user => 'anonymous'</font></div>
<div class=""><font face="Courier New" class="">);</font></div>
<div class=""><span class="Apple-tab-span" style="white-space:pre"><font face="Courier New" class=""></font></span></div>
<div class=""><font face="Courier New" class="">my $variation_adaptor = $registry->get_adaptor('homo_sapiens', 'variation', 'variation' );</font></div>
<div class=""><font face="Courier New" class="">my $ldfc_adaptor = $registry->get_adaptor('homo_sapiens', 'variation', 'ldfeaturecontainer');</font></div>
<div class=""><font face="Courier New" class="">my $population_adaptor = $registry->get_adaptor('homo_sapiens', 'variation', 'population');</font></div>
<div class=""><font face="Courier New" class="">$variation_adaptor->db->use_vcf(1); # To get 1000G phase 3 data also</font></div>
<div class=""><font face="Courier New" class=""><br class="">
</font></div>
<div class=""><font face="Courier New" class="">my $ld_populations = $population_adaptor->fetch_all_LD_Populations();</font></div>
<div class=""><font face="Courier New" class="">foreach my $ld_population (@$ld_populations) {</font></div>
<div class=""><font face="Courier New" class=""> print $ld_population->name, "\n";</font></div>
<div class=""><font face="Courier New" class="">}<span class="Apple-tab-span" style="white-space:pre">
</span></font></div>
<div class=""><font face="Courier New" class=""><br class="">
</font></div>
<div class=""><font face="Courier New" class="">my $variation_name = 'rs157580';</font></div>
<div class=""><font face="Courier New" class="">my $variation = $variation_adaptor->fetch_by_name($variation_name);</font></div>
<div class=""><font face="Courier New" class="">my @vfs = @{ $variation->get_all_VariationFeatures() };</font></div>
<div class=""><font face="Courier New" class=""><br class="">
</font></div>
<div class=""><font face="Courier New" class="">foreach my $vf (@vfs) {</font></div>
<div class=""><font face="Courier New" class=""><br class="">
</font></div>
<div class=""><font face="Courier New" class=""> print $vf->name, "\n";</font></div>
<div class=""><font face="Courier New" class=""> my @vf_pops = @{ $vf->get_all_LD_Populations() };</font></div>
<div class=""><font face="Courier New" class=""> foreach my $ld_population (@$ld_populations) {</font></div>
<div class=""><font face="Courier New" class=""> print $ld_population->name, "\n";</font></div>
<div class=""><font face="Courier New" class=""> my $ldfc = $ldfc_adaptor->fetch_by_VariationFeature($vf, $ld_population);</font></div>
<div class=""><font face="Courier New" class=""> foreach my $ld_hash (@{$ldfc->get_all_ld_values}) {</font></div>
<div class=""><font face="Courier New" class=""> <span class="Apple-tab-span" style="white-space:pre">
</span>my $d_prime = $ld_hash->{d_prime};</font></div>
<div class=""><font face="Courier New" class=""> <span class="Apple-tab-span" style="white-space:pre">
</span>my $r2 = $ld_hash->{r2};</font></div>
<div class=""><font face="Courier New" class=""><span class="Apple-tab-span" style="white-space:pre"></span>my $variation_name1 = $ld_hash->{variation1}->variation_name;</font></div>
<div class=""><font face="Courier New" class=""> <span class="Apple-tab-span" style="white-space:pre">
</span>my $variation_name2 = $ld_hash->{variation2}->variation_name;</font></div>
<div class=""><font face="Courier New" class=""> <span class="Apple-tab-span" style="white-space:pre">
</span>print "$variation_name1 $variation_name2 d_prime=$d_prime r2=$r2\n";</font></div>
<div class=""><font face="Courier New" class=""> }</font></div>
<div class=""><font face="Courier New" class=""> }</font></div>
<div class=""><font face="Courier New" class="">}</font></div>
<div class=""><font face="Courier New" class=""><br class="">
</font></div>
<div class=""><font face="Courier New" class="">my $end_run = time();</font></div>
<div class=""><font face="Courier New" class="">my $run_time = $end_run - $start_run;</font></div>
<div class=""><font face="Courier New" class="">print "Job took $run_time seconds\n";</font></div>
</div>
<div class=""><font face="Courier New" class=""><br class="">
</font></div>
<div class="">If I remove the call to get_all_LD_Populations, the script runs fine again. Do you have any idea on what I am doing wrong? Could it be a bug in the code, like the error I reported yesterday?</div>
<div class=""><br class="">
</div>
<div class="">Also, I have visited a lot of forums where LD calculation is discussed. Many users ask for a database one can query to find LD between SNPs, and genomic LD tracks, but all such services are only available on HapMap data. Do you know why there
is none for all SNPs and LD produced up until 1000G phase 3? What kind of restrictions is there that makes it easier to compute LD on the fly, for instance? Space maybe? </div>
<div class="">(If there actually does exist databases/tracks of LD, I would be happy to know!)</div>
<div class=""><br class="">
</div>
<div class="">Best,</div>
<div class="">Johanne</div>
<div class=""><br class="">
</div>
</div>
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