DoBlastzChainNet.pl: Difference between revisions
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screen -S dm6.GCF_000005575 | screen -S dm6.GCF_000005575 | ||
time (/data/scripts/ | time (/data/scripts/doBlastzChainNet.pl `pwd`/DEF -verbose=2 -noDbNameCheck \ | ||
-workhorse=localhost -bigClusterHub=localhost -skipDownload \ | -workhorse=localhost -bigClusterHub=localhost -skipDownload \ | ||
-dbHost=localhost -smallClusterHub=localhost \ | -dbHost=localhost -smallClusterHub=localhost \ | ||
Revision as of 15:14, 26 April 2018
Licensing
For commercial use of these toolsets, please note the license considerations for the kent source tools at the: Genome Store
Prerequisites
This discussion assumes you are familiar with Unix shell command line programming and scripting. You will be encountering and interacting with csh/tcsh, bash, perl, and python scripting languages. You will need at least one computer with several CPU cores, preferably a multiple compute cluster system or equivalent in a cloud computing environment.
This entire discussion assumes the bash shell is the user's unix shell.
Parasol Job Control System
The scripts and programs used here expect to find the Parasol_job_control_system in place and operational.
Install scripts and kent command line utilities
This is a bit of a kludge at this time (April 2018), we are working on a cleaner distribution of these scripts. As was mentioned in the Parasol_job_control_system setup, the kent command line binaries and these scripts are going to reside in /data/bin/ and /data/scripts/. This is merely a style custom to keep scripts separate from binaries, this is not strictly necessary to keep them separate.
mkdir -p /data/scripts /data/bin
chmod 755 /data/scripts /data/bin
rsync -a rsync://hgdownload.soe.ucsc.edu/genome/admin/exe/linux.x86_64/ /data/bin/
git archive --remote=git://genome-source.soe.ucsc.edu/kent.git \
--prefix=kent/ HEAD src/hg/utils/automation \
| tar vxf - -C /data/scripts --strip-components=5 \
--exclude='kent/src/hg/utils/automation/incidentDb' \
--exclude='kent/src/hg/utils/automation/configFiles' \
--exclude='kent/src/hg/utils/automation/ensGene' \
--exclude='kent/src/hg/utils/automation/genbank' \
--exclude='kent/src/hg/utils/automation/lastz_D' \
--exclude='kent/src/hg/utils/automation/openStack'
wget -O /data/bin/bedSingleCover.pl 'http://genome-source.soe.ucsc.edu/gitweb/?p=kent.git;a=blob_plain;f=src/utils/bedSingleCover.pl'
NOTE: A copy of the lastz binary is included in the rsync download of binaries from hgdownload. It is named lastz-1.04.00 to identify the version. Source for lastz can be obtained from lastz github.
PATH setup
Add or verify the two directories /data/bin and /data/scripts are added to the shell PATH environment. This can be added simply to the .bashrc file in your home directory:
echo 'export PATH=/data/bin:/data/scripts:$PATH' >> $HOME/.bashrc
Then, source that file to add that to this current shell:
. $HOME/.bashrc
Verify you see those pathnames on the PATH variable:
echo $PATH /data/bin:/data/scripts:/usr/local/bin:/usr/bin:/usr/local/sbin:/usr/sbin:/home/centos/.local/bin:/home/centos/bin
Working directory hierarchy
It is best to organize your work in a directory hierarchy. For example maintain all your genome sequences in:
/data/genomes/ /data/genomes/hg38/ /data/genomes/mm10/ /data/genomes/dm6/ /data/genomes/ce11/ ... etc ...
Where those database directories can have the 2bit files, chrom.sizes, and track construction directories, for example:
/data/genomes/dm6/dm6.2bit /data/genomes/dm6/dm6.chrom.sizes /data/genomes/dm6/trackData/
Such organizations are a personal preference custom. However you do this, keep it consistent to make it easier to use scripts on multiple sequences.
Obtain genome sequences
Genome sequences from the U.C. Santa Cruz Genomics Institute can be obtained directly from the hgdownload server via rsync. For example
mkdir /data/genomes/dm6 cd /data/genomes/dm6 rsync -avzP \ rsync://hgdownload.soe.ucsc.edu/goldenPath/dm6/bigZips/dm6.2bit . rsync -avzP \ rsync://hgdownload.soe.ucsc.edu/goldenPath/dm6/bigZips/dm6.chrom.sizes . ls -og -rw-rw-r--. 1 36969050 Aug 28 2014 dm6.2bit -rw-rw-r--. 1 45055 Aug 28 2014 dm6.chrom.sizes
Genome sequences from the NCBI/Entrez/Genbank system can be found via the assembly_summary.txt text listing information files, for example invertebrate genomes:
wget -O /tmp/invertebrate.assembly_summary.txt 'ftp://ftp.ncbi.nlm.nih.gov/genomes/refseq/invertebrate/assembly_summary.txt'
Looking for the Anopheles genome:
grep -w Anopheles /tmp/invertebrate.assembly_summary.txt GCF_000005575.2 PRJNA163 SAMN02952903 AAAB00000000.1 representative genome 180454 7165 Anopheles gambiae str. PEST strain=PEST latest Chromosome Major Full 2006/10/16 AgamP3 The International Consortium for the Sequencing of Anopheles Genome GCA_000005575.1 different ftp://ftp.ncbi.nlm.nih.gov/genomes/all/GCF/000/005/575/GCF_000005575.2_AgamP3
Note the Assembly identification from the ftp path GCF_000005575.2_AgamP3, working with that sequence, using the rsync service in place of the FTP:
mkdir /data/genomes/dm6/trackData/GCF_000005575.2_AgamP3
cd /data/genomes/dm6/trackData/GCF_000005575.2_AgamP3
rsync -L -a -P rsync://ftp.ncbi.nlm.nih.gov/genomes/all/GCF/000/005/575/GCF_000005575.2_AgamP3/GCF_000005575.2_AgamP3_genomic.fna.gz ./
rsync -L -a -P \
rsync://ftp.ncbi.nlm.nih.gov/genomes/all/GCF/000/005/575/GCF_000005575.2_AgamP3/GCF_000005575.2_AgamP3_assembly_report.txt ./
ls -og
total 79908
-rw-rw-r--. 1 811394 Mar 7 2017 GCF_000005575.2_AgamP3_assembly_report.txt
-rw-rw-r--. 1 81010275 Jun 15 2016 GCF_000005575.2_AgamP3_genomic.fna.gz
The assembly_report.txt file is useful to have for the meta-data information it has about the assembly. The fna.gz file needs to be in 2bit format for the processing system, and the chrom.sizes made from the 2bit:
faToTwoBit GCF_000005575.2_AgamP3_genomic.fna.gz GCF_000005575.2_AgamP3.2bit twoBitInfo GCF_000005575.2_AgamP3.2bit stdout | sort -k2,2nr > GCF_000005575.2_AgamP3.chrom.sizes ls -og total 156132 -rw-rw-r--. 1 77912208 Apr 6 03:48 GCF_000005575.2_AgamP3.2bit -rw-rw-r--. 1 138303 Apr 6 03:48 GCF_000005575.2_AgamP3.chrom.sizes -rw-rw-r--. 1 811394 Mar 7 2017 GCF_000005575.2_AgamP3_assembly_report.txt -rw-rw-r--. 1 81010275 Jun 15 2016 GCF_000005575.2_AgamP3_genomic.fna.gz
lastz parameter file
The DEF file is used with the script to specify alignment parameters to lastz and the axtChain operations. The example for dm6 target vs. A. gambiae query sequence, loose parameters are used for this distant alignment:
cat DEF # dm6 vs GCF_000005575.2_AgamP3 PATH=/data/bin:/data/scripts BLASTZ=/data/bin/lastz-1.04.00 BLASTZ_H=2000 BLASTZ_Y=3400 BLASTZ_L=4000 BLASTZ_K=2200 BLASTZ_Q=/data/lastz/HoxD55.q # TARGET: D. melanogaster dm6 SEQ1_DIR=/data/genomes/dm6/dm6.2bit SEQ1_LEN=/data/genomes/dm6/dm6.chrom.sizes SEQ1_CHUNK=32100000 SEQ1_LAP=10000 SEQ1_LIMIT=18 # QUERY: GCF_000005575.2_AgamP3 SEQ2_DIR=/data/genomes/dm6/trackData/GCF_000005575.2_AgamP3/GCF_000005575.2_AgamP3.2bit SEQ2_LEN=/data/genomes/dm6/trackData/GCF_000005575.2_AgamP3/GCF_000005575.2_AgamP3.chrom.sizes SEQ2_CHUNK=1000000 SEQ2_LIMIT=2000 SEQ2_LAP=0 BASE=/data/genomes/dm6/trackData/GCF_000005575.2_AgamP3 TMPDIR=/dev/shm
NOTE: UCSC tends to keep options for running alignments to approximately four category sets:
- human to other primates
- human to other mammals
- human to more distant vertebrates
- fly and worm alignments and other such distant organisms
Many examples of DEF files and chaining arguments can be found in the record of alignments at UCSC in the source tree make doc files. For example, alignments to human/hg38: hg38 lastz and the 100-way alignment: Hg38_100-way_conservation_lastz_parameters
Many experiments have been tried over time. To keep it simple:
- human to other primates
BLASTZ_M=254 BLASTZ_O=600 BLASTZ_E=150 BLASTZ_K=4500 BLASTZ_Y=15000 BLASTZ_T=2 BLASTZ_Q=/scratch/data/blastz/human_chimp.v2.q # Where human_chimp.v2.q is: # A C G T # 90 -330 -236 -356 # -330 100 -318 -236 # -236 -318 100 -330 # -356 -236 -330 90 -chainMinScore=5000 -chainLinearGap=medium
- human to other mammals
BLASTZ_O=400 BLASTZ_E=30 BLASTZ_M=254 # default BLASTZ_Q score matrix: # A C G T # A 91 -114 -31 -123 # C -114 100 -125 -31 # G -31 -125 100 -114 # T -123 -31 -114 91 -chainMinScore=3000 -chainLinearGap=medium
- human to more distant vertebrates
BLASTZ_M=50 BLASTZ_Y=3400 BLASTZ_L=6000 BLASTZ_K=2200 BLASTZ_Q=/scratch/data/blastz/HoxD55.q # HoxD55.q matrix is: # A C G T # 91 -90 -25 -100 # -90 100 -100 -25 # -25 -100 100 -90 # -100 -25 -90 91 -chainMinScore=5000 -chainLinearGap=loose
- fly and worm alignments and other such distant organisms
BLASTZ_H=2000 BLASTZ_Y=3400 BLASTZ_L=4000 BLASTZ_K=2200 BLASTZ_Q=/data/lastz/HoxD55.q # HoxD55.q matrix is: # A C G T # 91 -90 -25 -100 # -90 100 -100 -25 # -25 -100 100 -90 # -100 -25 -90 91 -chainMinScore=1000 -chainLinearGap=loose
perform alignment
After the DEF file is established, verify the files specified in it are actually present at the locations specified:
egrep "_DIR|_LEN" DEF | sed -e 's/.*=//;' | xargs ls -og -rw-rw-r--. 1 36969050 Aug 28 2014 /data/genomes/dm6/dm6.2bit -rw-rw-r--. 1 45055 Aug 28 2014 /data/genomes/dm6/dm6.chrom.sizes -rw-rw-r--. 1 77912208 Apr 6 03:48 /data/genomes/dm6/trackData/GCF_000005575.2_AgamP3/GCF_000005575.2_AgamP3.2bit -rw-rw-r--. 1 138303 Apr 6 03:48 /data/genomes/dm6/trackData/GCF_000005575.2_AgamP3/GCF_000005575.2_AgamP3.chrom.sizes
Use a screen to keep this command attached to a terminal that you can detach from and reattach to at a later time. For large genomes, or with fewer CPU cores available, this command can run for many days. Time the operation of the command and record all output from it for later analysis if any problems arise from the operation:
screen -S dm6.GCF_000005575
time (/data/scripts/doBlastzChainNet.pl `pwd`/DEF -verbose=2 -noDbNameCheck \
-workhorse=localhost -bigClusterHub=localhost -skipDownload \
-dbHost=localhost -smallClusterHub=localhost \
-trackHub -fileServer=localhost -syntenicNet) > do.log 2>&1 &
The screen -S dm6.GCF_000005575 gives a name to the terminal so you can find it later in a listing of a number of screens. To detach from the running terminal, use two key presses:
"Ctrl-a Ctrl-d"
to reattach to this screen: screen -r -d dm6.GCF_000005575
BEWARE the drawback of the screen is that you can accidentally exit the shell while in the screen and you thus lose it. The processes that were attached to that shell can continue if they do not respond to the SIGHUP signal. To avoid this side-effect, develop a habit of always exiting a shell with the two key presses "Ctrl-a Ctrl-d", in a shell that is not in a screen it will merely echo those keystrokes and do nothing.
Monitor progress
To determine which step the process is working on, in this working directory, look for the word step in the do.log file:
cd /data/genomes/dm6/trackData/GCF_000005575.2_AgamP3 grep -w step do.log HgStepManager: executing from step 'partition' through step 'syntenicNet'. HgStepManager: executing step 'partition' Fri Apr 6 04:50:34 2018. HgStepManager: executing step 'blastz' Fri Apr 6 04:50:46 2018.
To view the parasol status of your batch:
parasol list batches #user run wait done crash pri max cpu ram plan min batch centos 323 18446 10283 0 10 -1 1 2.0g 323 0 /data/genomes/dm6/trackData/GCF_000005575.2_AgamP3/run.blastz/
To view the status of that particular batch:
cd /data/genomes/dm6/trackData/GCF_000005575.2_AgamP3/run.blastz/ para time 29052 jobs in batch 16647 jobs (including everybody's) in Parasol queue or running. Checking finished jobs .......................... Completed: 12405 of 29052 jobs Jobs currently running: 323 In queue waiting: 16324 jobs CPU time in finished jobs: 81145s 1352.42m 22.54h 0.94d 0.003 y IO & Wait Time: 142817s 2380.28m 39.67h 1.65d 0.005 y Time in running jobs: 4076s 67.93m 1.13h 0.05d 0.000 y Average job time: 18s 0.30m 0.01h 0.00d Longest running job: 43s 0.72m 0.01h 0.00d Longest finished job: 72s 1.20m 0.02h 0.00d Submission to last job: 718s 11.97m 0.20h 0.01d Estimated complete: 930s 15.51m 0.26h 0.01d
This example happens to be running on an Open Stack cluster with 323 allocated CPU cores:
parasol status CPUs total: 323 CPUs free: 0 CPUs busy: 323 Nodes total: 20 Nodes dead: 0 Nodes sick?: 0 Jobs running: 323 Jobs waiting: 14977 Jobs finished: 13752 Jobs crashed: 0 Spokes free: 30 Spokes busy: 0 Spokes dead: 0 Active batches: 1 Total batches: 1 Active users: 1 Total users: 1 Days up: 0.012685 Version: 12.18
When a parasol batch is completed, this scripting process leaves a run.time file in the batch directory where you can see what type of cluster time you have used:
cd /data/genomes/dm6/trackData/GCF_000005575.2_AgamP3/run.blastz cat run.time Completed: 29052 of 29052 jobs CPU time in finished jobs: 149061s 2484.35m 41.41h 1.73d 0.005 y IO & Wait Time: 268812s 4480.20m 74.67h 3.11d 0.009 y Average job time: 14s 0.24m 0.00h 0.00d Longest finished job: 72s 1.20m 0.02h 0.00d Submission to last job: 1312s 21.87m 0.36h 0.02d Estimated complete: 0s 0.00m 0.00h 0.00d
For small genomes such as the two in this example, the steps after the lastz alignment can proceed rapidly:
grep -w step do.log HgStepManager: executing from step 'partition' through step 'syntenicNet'. HgStepManager: executing step 'partition' Fri Apr 6 04:50:34 2018. HgStepManager: executing step 'blastz' Fri Apr 6 04:50:46 2018. HgStepManager: executing step 'cat' Fri Apr 6 05:13:52 2018. HgStepManager: executing step 'chainRun' Fri Apr 6 05:14:12 2018. HgStepManager: executing step 'chainMerge' Fri Apr 6 05:15:49 2018. HgStepManager: executing step 'net' Fri Apr 6 05:15:58 2018. HgStepManager: executing step 'load' Fri Apr 6 05:17:06 2018. HgStepManager: executing step 'download' Fri Apr 6 05:17:35 2018. HgStepManager: executing step 'cleanup' Fri Apr 6 05:17:36 2018. HgStepManager: executing step 'syntenicNet' Fri Apr 6 05:17:42 2018.
The syntenicNet is the last step in this process, the do.log timing will indicate the full time for this alignment:
tail -3 do.log real 27m20.456s user 0m0.720s sys 0m0.394s
And featureBits measurements have taken place to indicate the amount of coverage of the target genome by the query genome, for both the fundamental alignment, and the syntenic filtered alignment:
ls fb.* fb.dm6.chain.GCF_000005575.2_AgamP3Link.txt fb.dm6.chainSyn.GCF_000005575.2_AgamP3Link.txt
cat fb.* 19155294 bases of 143726002 (13.328%) in intersection 1617815 bases of 143726002 (1.126%) in intersection
Reciprocal Best
After that alignment is completed, the reciprocal best alignment can be computed:
cd /data/genomes/dm6/trackData/GCF_000005575.2_AgamP3
export tDb=`grep "SEQ1_DIR=" DEF | sed -e 's#.*/##; s#.2bit##;'`
export qDb=`grep "SEQ2_DIR=" DEF | sed -e 's#.*/##; s#.2bit##;'`
export target2Bit=`grep "SEQ1_DIR=" DEF | sed -e 's/.*=//;'`
export targetSizes=`grep "SEQ1_LEN=" DEF | sed -e 's/.*=//;'`
export query2Bit=`grep "SEQ2_DIR=" DEF | sed -e 's/.*=//;'`
export querySizes=`grep "SEQ2_LEN=" DEF | sed -e 's/.*=//;'`
time (/data/scripts/doRecipBest.pl -buildDir=`pwd` -load \
-workhorse=localhost -dbHost=localhost -skipDownload \
-target2Bit=${target2Bit} -query2Bit=${query2Bit} \
-targetSizes=${targetSizes} -querySizes=${querySizes} \
-trackHub ${tDb} ${qDb}) > rbest.log 2>&1 &
This process does not have any parasol batch procedures. The procedure only does transformations on some of the result files computed during the first alignment procedure. Since this is not a parallel procedure, it takes a bit of time:
grep -w step rbest.log HgStepManager: executing from step 'recipBest' through step 'cleanup'. HgStepManager: executing step 'recipBest' Fri Apr 6 05:24:27 2018. HgStepManager: executing step 'download' Fri Apr 6 05:55:38 2018. HgStepManager: executing step 'load' Fri Apr 6 05:55:38 2018. HgStepManager: executing step 'cleanup' Fri Apr 6 05:55:47 2018.
When completed, this has a featureBits measurement also:
cat fb.dm6.chainRBest.GCF_000005575.2_AgamP3.txt 15316412 bases of 143726002 (10.657%) in intersection
Track Hub files
This procedure has constructed big* files that can be used to display these tracks in a track hub on the UCSC genome browser
ls axtChain/*.bb bigMaf/*.bb axtChain/chainGCF_000005575.2_AgamP3.bb axtChain/chainGCF_000005575.2_AgamP3Link.bb axtChain/chainRBestGCF_000005575.2_AgamP3.bb axtChain/chainRBestGCF_000005575.2_AgamP3Link.bb axtChain/chainSynGCF_000005575.2_AgamP3.bb axtChain/chainSynGCF_000005575.2_AgamP3Link.bb bigMaf/dm6.GCF_000005575.2_AgamP3.net.bb bigMaf/dm6.GCF_000005575.2_AgamP3.net.summary.bb bigMaf/dm6.GCF_000005575.2_AgamP3.rbestNet.bb bigMaf/dm6.GCF_000005575.2_AgamP3.rbestNet.summary.bb bigMaf/dm6.GCF_000005575.2_AgamP3.synNet.bb bigMaf/dm6.GCF_000005575.2_AgamP3.synNet.summary.bb
(TBD: show structure of trackDb.txt track hub specifications)
How does this process work
The doBlastzChainNet.pl script performs the processing in distinct steps. Each step is almost always performed with a C-shell or bash shell script. Therefore, if there is a problem in any step, the commands performing the step can be dissected from the script in operation, the problem identified and fixed, and the step completed manually by running the rest of the commands in that script. Once a step has been completed, the process can continue with the next step using the argument -continue=nextStepName. Check the usage message from the doBlastzChainNet.pl script to see a listing of the steps and their sequence. Specifically:
partition, blastz, cat, chainRun, chainMerge, net, load, download, cleanup, syntenicNet
In this example, the various scripts are:
-rwxrwxr-x. 1 1914 Apr 6 04:50 run.blastz/doPartition.bash -rw-rw-r--. 1 2713 Apr 6 04:50 run.blastz/xdir.sh -rwxrwxr-x. 1 606 Apr 6 04:50 run.blastz/doClusterRun.csh -rwxrwxr-x. 1 802 Apr 6 05:13 run.cat/doCatRun.csh -rwxrwxr-x. 1 72 Apr 6 05:13 run.cat/cat.csh -rwxrwxr-x. 1 412 Apr 6 05:14 axtChain/run/chain.csh -rwxrwxr-x. 1 700 Apr 6 05:14 axtChain/run/doChainRun.csh -rwxrwxr-x. 1 3112 Apr 6 05:15 axtChain/netChains.csh -rwxrwxr-x. 1 2162 Apr 6 05:17 axtChain/loadUp.csh -rwxrwxr-x. 1 1479 Apr 6 05:17 cleanUp.csh -rwxrwxr-x. 1 4507 Apr 6 05:17 axtChain/netSynteny.csh -rwxrwxr-x. 1 6321 Apr 6 05:24 axtChain/doRecipBest.csh -rwxrwxr-x. 1 1974 Apr 6 05:55 axtChain/loadRBest.csh -rwxrwxr-x. 1 583 Apr 6 05:55 rBestCleanUp.bash
