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--- contributors:team_4_page [2015/05/08 02:52]
JaredC
+++ contributors:team_4_page [2015/07/18 20:52] (current)
92.247.181.31 ↷ Links adapted because of a move operation
@@ Line 20: / Line 20: @@
 [[https://github.com/bcgsc/abyss| ABySS manual and source code]]
+=====General notes=====
+  - ABySS can run in serial mode, but that isn't too useful for such a large genome.
+  - The documentation recommends creating assemblies with several values of k and selecting the "best" one.
+  - The program involves its own error correction.
 =====Installing ABySS=====
@@ Line 34: / Line 39: @@
 % --enable-dependency-tracking\
 % --with-boost=/campusdata/BME235/include/boost\
-% --with-mpi=/campusdata/BME235/include\
+% --with-mpi=/opt/openmpi\
-% CC=gcc-4.9.2 CXX=g++-4.9.2\
+% CC=gcc CXX=g++\
-% CPPFLAGS=-I/campusdata/BME235/include/sparsehash
+% CPPFLAGS=-I/campusdata/BME235/include/
 </code>
 Then ABySS can be installed via the makefile
@@ Line 43: / Line 48: @@
 % make install
 </code>
+==== Notes on Installation ====
+  * the //-enable-maxk// is by default set to 64. Always ensure the max kmer is set to the smallest possible value that accommodates your target kmers so as to minimize ABySS's memory footprint.
+  * ensure that the CPPFLAGS specified directory holds the google sparsehash includes. Look for a warning in the ABySS compilation to be sure.
+  * the boost libraries do not need to be compiled before installing.
+  * ensure openmpi is installed with the --with-sge option (for SGE) to ensure tight integration.
 =====ABySS parameters=====
@@ Line 75: / Line 85: @@
 =====Running ABySS=====
+Running an assembly on the campusrocks cluster, which runs on SGE, requires the use of the qsub command.  The use of a shell script allows a convenient and concise way to wrap useful qsub options, environmental variable manipulations, and the executable (abyss-pe) itself in a single script. An example script which runs
+the parallel-version of ABySS on the cluster is shown below.
+<code>
+#!/bin/sh
+#$ -N team4
+#$ -cwd
+#$ -j y
+#$ -pe mpi 10
+#$ -S /bin/bash
+#$ -V
+ABYSSRUNDIR=/campusdata/BME235/bin
+export PATH=$PATH:/opt/openmpi/bin:/campusdata/BME235/bin/
+export LD_LIBRARY_PATH=/opt/openmpi/lib/:$LD_LIBRARY_PATH
+ABYSSRUN=$ABYSSRUNDIR/abyss-pe
+$ABYSSRUN np=10 k=21 name=ecoli in='/campusdata/BME235/programs/abyss-1.5.2/JARED/test-data/reads1.fastq /campusdata/BME235/programs/abyss-1.5.2/JARED/test-data/reads2.fastq'
+</code>
+Note that the parallel version of ABySS requires two things in particular:
+  * The use of a [[archive:parallel_environment]] (PE) which can be selected using a qsub option.
+  * The //np// option of abyss-pe. The number of processes here must reflect the number included in the parallel environment option.
+The PE option in the script above:
+<code>
+#$ -pe mpi 10
+</code>
+The //mpi// designates the choice of a PE that is installed on the system and the 10 indicates the number of processes over which to run the job. To see which PE's are installed on the system, use the command:
+<code>
+qconf -spl
+</code>
+Selecting the proper PE is critical to ensure the success of a parallelized ABySS job.  By using the command:
+<code>
+qconf -sp PE_NAME
+</code>
+you can inspect the settings for an individual PE.  For example, using the command //qconf -sp mpi// will report the following:
+<code>
+pe_name            mpi
+slots              9999
+user_lists         NONE
+xuser_lists        NONE
+start_proc_args    /opt/gridengine/mpi/startmpi.sh $pe_hostfile
+stop_proc_args     /opt/gridengine/mpi/stopmpi.sh
+allocation_rule    $fill_up
+control_slaves     FALSE
+job_is_first_task  TRUE
+urgency_slots      min
+accounting_summary TRUE
+</code>
+For using ABySS with openmpi, there are three settings in particular which should be noted:
+  * slots: indicates maximum number of slots that can be designated with the PE
+  * allocation_rule: indicates the form of scheduling to be used to allocate slots to nodes
+  * control_slaves: used to indicate a tightly managed interface
+For all of the PE's installed on the campus cluster, the slots were all set at 9999 (virtually limitless) and so were not of terrible concern to us. However, the allocation_rule and control_slaves were critical. The initial PE tried was //mpi// however, because openmpi can achieve tight integration with the cluster's SGE, we switched to orte. This was found to be inadequate due to its allocation_rule of $pe_slots. This demanded that all slots requested by the job be accommodated on a single node. This proved to be fatal since the memory requirements of a run with full libraries required more memory than that possessed by the largest node (~256 GB). We then switched to mpich which offers both tight integration and an allocation_rule of $fill_up which, when used with a sufficiently large number of slots, would ensure that multiple nodes are used in a job. However, the limitation in this setting is that, depending on the relative traffic on the cluster, we would have to designate more slots than needed to ensure the use of multiple nodes.  To address this, we had the //denovo// PE created and added to the queue.  This PE offers both tight integration and an allocation_rule of $round_robin.  This allocation setting ensures that the slots requested are evenly distributed across available nodes.  Thus, selecting relatively few processes would ensure the use of multiple nodes' memory.
 abyss-pe is a driver script implemented as a Makefile. Any option of make may be used with abyss-pe. Particularly useful options are:
@@ Line 133: / Line 194: @@
   * konnector: fill the gaps between paired-end reads by building a Bloom filter de Bruijn graph and searching for paths between paired-end reads within the graph
   * abyss-bloom: construct reusable bloom filter files for input to Konnector
 =====ABySS pipeline=====
 {{ :bioinformatic_tools:abysspipeline.png?nolink |}}
+=====Test run=====
+This run was done using version 1.5.2. The assembly used k=59, 10 processes, and requested mem_free=15g from qsub. The assembly was done using the SW018 and SW019 libraries only. Specifically, the files used were:
+  * /campusdata/BME235/Spring2015Data/adapter_trimming/SeqPrep/SW018_S1_L007_R1_001_trimmed.fastq.gz
+  * /campusdata/BME235/Spring2015Data/adapter_trimming/SeqPrep/SW018_S1_L007_R2_001_trimmed.fastq.gz
+  * /campusdata/BME235/Spring2015Data/adapter_trimming/SeqPrep/SW019_S1_L001_R1_001_trimmed.fastq.gz
+  * /campusdata/BME235/Spring2015Data/adapter_trimming/SeqPrep/SW019_S1_L001_R2_001_trimmed.fastq.gz
+  * /campusdata/BME235/Spring2015Data/adapter_trimming/SeqPrep/SW019_S2_L008_R1_001_trimmed.fastq.gz
+  * /campusdata/BME235/Spring2015Data/adapter_trimming/SeqPrep/SW019_S2_L008_R2_001_trimmed.fastq.gz
+The files had adapters trimmed using SeqPrep (see the data pages for more details). SW019_S1 and SW019_S2 were treated as two separate libraries.
+The output and log files for this assembly are in /campusdata/BME235/S15_assemblies/abyss/sidra/test_run/singleK.
+====Results====
+Note: the N50, etc., stats only include contigs >= 500 bp (I believe the rest are discarded).
+There are 10.23 * 10^6 contigs. The N50 contig size is 2,669. The number of contigs of at least N50 (n:N50) is 174,507. The maximum contig size is 31,605, and the total number of bp (in contigs >= 500 bp) is 1.557 * 10^9.
+Here are the stats summarized for the contigs and also for scaffolds and unitigs. n:500 is the number of contigs/unitigs/scaffolds at least as long as 500 bp. sum is the number of bases in all the contigs/unitigs/scaffolds at least as long as 500 bp combined.
+| n | n:500 | n:N50 | min | N80 | N50 | N20 | E-size | max | sum | name |
+| 11.95e6 | 993409 | 247109 | 500 | 962  | 1795 | 3327 | 2296 | 30520 | 1.456e9 | slug-unitigs.fa |
+| 10.23e6 | 785054 | 174507 | 500 | 1320 | 2669 | 5079 | 3433 | 31605 | 1.557e9 | slug-contigs.fa |
+| 10.11e6 | 711022 | 153036 | 500 | 1490 | 3063 | 5870 | 3945 | 37466 | 1.573e9 | slug-scaffolds.fa |
+====Notes====
+The success of this run means we are probably ready to do a run with all the data (not including the mate-pair data, that can be used for scaffolding later). For that run, the different trimmed files for each library should be concatenated, so that the run involves only the actual number of libraries we had (I believe 4?). It should also use many more than 10 processes.
+=====Test with more data=====
+This run was also done using version 1.5.2. I used SGE job arrays to submit multiple assemblies with different k values, k=55-65 and 27-37, odds only. (Using this many similar ks is probably unnecessary.) The syntax for submitting an array w/ multiple ks is
+  qsub -t 55-65:2 slug-pe.sh
+where slug-pe.sh uses $SGE_TASK_ID in place of the k value. I used 32 processes per job and tried submitting with various amounts of memory requested, from 10-30g. I also added a quality cutoff of 20 (parameter q=20) to this run. The files used this time were concatenated versions of the various sequencing runs for each library:
+  * /campusdata/BME235/Spring2015Data/adapter_trimming/SeqPrep_catted_libs/SW018_S1_and_UCSF_R1_trimmed.fastq.gz
+  * /campusdata/BME235/Spring2015Data/adapter_trimming/SeqPrep_catted_libs/SW018_S1_and_UCSF_R2_trimmed.fastq.gz
+  * /campusdata/BME235/Spring2015Data/adapter_trimming/SeqPrep_catted_libs/SW019_S1_S2_and_UCSF_R1_trimmed.fastq.gz
+  * /campusdata/BME235/Spring2015Data/adapter_trimming/SeqPrep_catted_libs/SW019_S1_S2_and_UCSF_R2_trimmed.fastq.gz
+  * /campusdata/BME235/Spring2015Data/adapter_trimming/SeqPrep_newData/UCSF_BS-MK_CATCCGG_R1_trimmed.fastq.gz
+  * /campusdata/BME235/Spring2015Data/adapter_trimming/SeqPrep_newData/UCSF_BS-MK_CATCCGG_R2_trimmed.fastq.gz
+  * /campusdata/BME235/Spring2015Data/adapter_trimming/SeqPrep_newData/UCSF_BS-tag_GTAGAGG_R1_trimmed.fastq.gz
+  * /campusdata/BME235/Spring2015Data/adapter_trimming/SeqPrep_newData/UCSF_BS-tag_GTAGAGG_R2_trimmed.fastq.gz
+The files had adapters trimmed using SeqPrep, but were not merged (see the data pages for more details). The four libraries used were SW018, SW019, BS-MK, and BS-tag.
+I was not able to get these to run successfully. On the first attempt, the job with k=55 started running and died after 6 hours. It was difficult to tell why based on the error (probably ran out of memory) so after this I added the v=-v (verbose) option. Here's the error I got (before verbose option):
+<code>
+/opt/openmpi/bin/mpirun -np 32 ABYSS-P -k55 -q20   --coverage-hist=coverage.hist -s slug-bubbles.fa  -
+o slug-1.fa /campusdata/BME235/Spring2015Data/adapter_trimming/SeqPrep_catted_libs/SW018_S1_and_UCSF_R
+_trimmed.fastq.gz /campusdata/BME235/Spring2015Data/adapter_trimming/SeqPrep_catted_libs/SW018_S1_and
+_UCSF_R2_trimmed.fastq.gz /campusdata/BME235/Spring2015Data/adapter_trimming/SeqPrep_catted_libs/SW019
+_S1_S2_and_UCSF_R1_trimmed.fastq.gz /campusdata/BME235/Spring2015Data/adapter_trimming/SeqPrep_catted_
+libs/SW019_S1_S2_and_UCSF_R2_trimmed.fastq.gz /campusdata/BME235/Spring2015Data/adapter_trimming/SeqPr
+ep_newData/UCSF_BS-MK_CATCCGG_R1_trimmed.fastq.gz /campusdata/BME235/Spring2015Data/adapter_trimming/S
+eqPrep_newData/UCSF_BS-MK_CATCCGG_R2_trimmed.fastq.gz /campusdata/BME235/Spring2015Data/adapter_trimmi
+ng/SeqPrep_newData/UCSF_BS-tag_GTAGAGG_R1_trimmed.fastq.gz /campusdata/BME235/Spring2015Data/adapter_t
+rimming/SeqPrep_newData/UCSF_BS-tag_GTAGAGG_R2_trimmed.fastq.gz
+ABySS 1.5.2
+ABYSS-P -k55 -q20 --coverage-hist=coverage.hist -s slug-bubbles.fa -o slug-1.fa /campusdata/BME235/Spr
+ing2015Data/adapter_trimming/SeqPrep_catted_libs/SW018_S1_and_UCSF_R1_trimmed.fastq.gz /campusdata/BME
+/Spring2015Data/adapter_trimming/SeqPrep_catted_libs/SW018_S1_and_UCSF_R2_trimmed.fastq.gz /campusdata/BME235/Spring2015Data/adapter_trimming/SeqPrep_catted_libs/SW019_S1_S2_and_UCSF_R1_trimmed.fastq.gz /campusdata/BME235/Spring2015Data/adapter_trimming/SeqPrep_catted_libs/SW019_S1_S2_and_UCSF_R2_trimmed.fastq.gz /campusdata/BME235/Spring2015Data/adapter_trimming/SeqPrep_newData/UCSF_BS-MK_CATCCGG_R1_trimmed.fastq.gz /campusdata/BME235/Spring2015Data/adapter_trimming/SeqPrep_newData/UCSF_BS-MK_CATCCGG_R2_trimmed.fastq.gz /campusdata/BME235/Spring2015Data/adapter_trimming/SeqPrep_newData/UCSF_BS-tag_GTAGAGG_R1_trimmed.fastq.gz /campusdata/BME235/Spring2015Data/adapter_trimming/SeqPrep_newData/UCSF_BS-tag_GTAGAGG_R2_trimmed.fastq.gz
+Running on 32 processors
+: Running on host campusrocks2-0-2.local
+: Running on host campusrocks2-0-2.local
+: Running on host campusrocks2-0-2.local
+: Running on host campusrocks2-0-2.local
+: Running on host campusrocks2-0-2.local
+: Running on host campusrocks2-0-2.local
+: Running on host campusrocks2-0-2.local
+: Running on host campusrocks2-0-2.local
+: Running on host campusrocks2-0-2.local
+: Running on host campusrocks2-0-2.local
+: Running on host campusrocks2-0-2.local
+: Running on host campusrocks2-0-2.local
+: Running on host campusrocks2-0-2.local
+: Running on host campusrocks2-0-2.local
+: Running on host campusrocks2-0-2.local
+: Running on host campusrocks2-0-2.local
+: Running on host campusrocks2-0-2.local
+: Running on host campusrocks2-0-2.local
+: Running on host campusrocks2-0-2.local
+: Running on host campusrocks2-0-2.local
+: Running on host campusrocks2-0-2.local
+: Running on host campusrocks2-0-2.local
+: Running on host campusrocks2-0-2.local
+: Running on host campusrocks2-0-2.local
+: Running on host campusrocks2-0-2.local
+: Running on host campusrocks2-0-2.local
+: Running on host campusrocks2-0-2.local
+: Running on host campusrocks2-0-2.local
+: Running on host campusrocks2-0-2.local
+: Running on host campusrocks2-0-2.local
+: Running on host campusrocks2-0-2.local
+: Running on host campusrocks2-0-2.local
+: Reading `/campusdata/BME235/Spring2015Data/adapter_trimming/SeqPrep_catted_libs/SW018_S1_and_UCSF_R1_trimmed.fastq.gz'...
+: Reading `/campusdata/BME235/Spring2015Data/adapter_trimming/SeqPrep_catted_libs/SW019_S1_S2_and_UCSF_R1_trimmed.fastq.gz'...
+: Reading `/campusdata/BME235/Spring2015Data/adapter_trimming/SeqPrep_catted_libs/SW019_S1_S2_and_UCSF_R2_trimmed.fastq.gz'...
+: Reading `/campusdata/BME235/Spring2015Data/adapter_trimming/SeqPrep_newData/UCSF_BS-tag_GTAGAGG_R2_trimmed.fastq.gz'...
+: Reading `/campusdata/BME235/Spring2015Data/adapter_trimming/SeqPrep_newData/UCSF_BS-MK_CATCCGG_R1_trimmed.fastq.gz'...
+: Reading `/campusdata/BME235/Spring2015Data/adapter_trimming/SeqPrep_catted_libs/SW018_S1_and_UCSF_R2_trimmed.fastq.gz'...
+: Reading `/campusdata/BME235/Spring2015Data/adapter_trimming/SeqPrep_newData/UCSF_BS-MK_CATCCGG_R2_trimmed.fastq.gz'...
+: Reading `/campusdata/BME235/Spring2015Data/adapter_trimming/SeqPrep_newData/UCSF_BS-tag_GTAGAGG_R1_trimmed.fastq.gz'...
+--------------------------------------------------------------------------
+mpirun noticed that process rank 0 with PID 8806 on node campusrocks2-0-2.local exited on signal 9 (Killed).
+--------------------------------------------------------------------------
+make: *** [slug-1.fa] Error 137
+</code>
+The job ran for about 6 hours before being killed, according to qacct.
+For all other jobs in the array, and for other tries using the same submission script, the jobs never started running, and I got this error:
+<code>/opt/openmpi/bin/mpirun -np 32 ABYSS-P -k57 -q20   --coverage-hist=coverage.hist -s slug-bubbles.fa  -o slug-1.fa /campusdata/BME235/Spring2015Data/adapter_trimming/SeqPrep_catted_libs/SW018_S1_and_UCSF_R1_trimmed.fastq.gz /campusdata/BME235/Spring2015Data/adapter_trimming/SeqPrep_catted_libs/SW018_S1_and_UCSF_R2_trimmed.fastq.gz /campusdata/BME235/Spring2015Data/adapter_trimming/SeqPrep_catted_libs/SW019_S1_S2_and_UCSF_R1_trimmed.fastq.gz /campusdata/BME235/Spring2015Data/adapter_trimming/SeqPrep_catted_libs/SW019_S1_S2_and_UCSF_R2_trimmed.fastq.gz /campusdata/BME235/Spring2015Data/adapter_trimming/SeqPrep_newData/UCSF_BS-MK_CATCCGG_R1_trimmed.fastq.gz /campusdata/BME235/Spring2015Data/adapter_trimming/SeqPrep_newData/UCSF_BS-MK_CATCCGG_R2_trimmed.fastq.gz /campusdata/BME235/Spring2015Data/adapter_trimming/SeqPrep_newData/UCSF_BS-tag_GTAGAGG_R1_trimmed.fastq.gz /campusdata/BME235/Spring2015Data/adapter_trimming/SeqPrep_newData/UCSF_BS-tag_GTAGAGG_R2_trimmed.fastq.gz
+error: executing task of job 81112 failed: execution daemon on host "campusrocks2-0-1.local" didn't accept task
+--------------------------------------------------------------------------
+A daemon (pid 43664) died unexpectedly with status 1 while attempting
+to launch so we are aborting.
+There may be more information reported by the environment (see above).
+This may be because the daemon was unable to find all the needed shared
+libraries on the remote node. You may set your LD_LIBRARY_PATH to have the
+location of the shared libraries on the remote nodes and this will
+automatically be forwarded to the remote nodes.
+--------------------------------------------------------------------------
+--------------------------------------------------------------------------
+mpirun noticed that the job aborted, but has no info as to the process
+that caused that situation.
+--------------------------------------------------------------------------
+--------------------------------------------------------------------------
+mpirun was unable to cleanly terminate the daemons on the nodes shown
+below. Additional manual cleanup may be required - please refer to
+the "orte-clean" tool for assistance.
+--------------------------------------------------------------------------
+        campusrocks2-0-1.local - daemon did not report back when launched
+        campusrocks2-0-2.local - daemon did not report back when launched
+make: *** [slug-1.fa] Error 1
+</code>
+It seems like the ABySS group in a previous iteration of the class had the same issue. I submitted an IT ticket and received absolutely no help. The only thing I could figure out is that this is some sort of issue with openmpi, so our next steps will be attempting the assembly using orte instead of openmpi and also trying the assembly on edser2. We are also going to try some end trimming first so that we have a better chance of not using up all the available memory.
+The output and log files for this assembly are in /campusdata/BME235/S15_assemblies/abyss/sidra/assembly and /campusdata/BME235/S15_assemblies/abyss/sidra/assembly/failed_runs.

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