Tag Archives: Ostrea lurida

BS-seq Mapping – Olympia oyster bisulfite sequencing: Bismark Continued

Previously took the analysis just through the mapping, but didn’t realize Steven wanted me to fully process the data.

So, as en exercise, I followed through with deduplication and sorting of the BAM files.

Then, ran a quick analysis using MethylKit in R. The analysis simply copied what Steven had done with another data set and I haven’t examined it very thoroughly, so am not well-versed on what it’s doing and/or why.

Jupyter Notebook (GitHub):

R Studio Project (download the folder, load project in R Studio, and then run the script in the scripts subdirectory to run the analysis):

Will take the full data sets through this whole pipeline.

Transposable Element Mapping – Olympia Oyster Genome Assembly using RepeatMasker 4.07

Steven wanted transposable elements (TEs) in the Olympia oyster genome identified.

After some minor struggles, I was able to get RepeatMasker installed on on both of our Apple Xserves (emu & roadrunner; running Ubuntu 16.04LTS).

Genome used: pbjelly_sjw_01

I ran RepeatMasker (v4.07) with RepBase-20170127 and RMBlast 2.6.0 four times:

  1. Default settings (i.e. no species select – will use human genome).

  2. Species = Crassostrea gigas (Pacific oyster)

  3. Species = Crassostrea virginica (Eastern oyster)

  4. Species = Ostrea lurida (Olympia oyster)

The idea was to get a sense of how the analyses would differ with species specifications. However, it’s likely that the only species setting that will make any difference will be Run #2 (Crassostrea gigas).

The reason I say this is that RepeatMasker has a built in tool to query which species are available in the RepBase database (e.g.):

RepeatMasker-4.0.7/util/queryRepeatDatabase.pl -species "crassostrea virginica" -stat

Here’s a very brief overview of what that yields:

  • Crassotrea gigas: 792 specific repeats

  • Crassostrea virginica: 4 Crassostrea virginica specific repeats

  • Ostrea lurida: 0 Ostrea lurida specific repeats

All runs were performed on roadrunner.

All commands were documented in a Jupyter Notebook (GitHub):

NOTE: RepeatMasker writes the desired output files (*.out, *.cat.gz, and *.gff) to the same directory that the genome is located in! If you conduct multiple runs with the same genome in the same directory, it will overwrite those files, as they are named using the genome assembly filename.


RESULTS:
RUN 1 (default settings – human genome)

Output folder:

Summary table (text):

Output table (GFF):

SUMMARY TABLE

==================================================
file name: jelly.out.fasta          
sequences:        696946
total length: 1253001795 bp  (1172226648 bp excl N/X-runs)
GC level:         36.51 %
bases masked:   20002806 bp ( 1.71 %)
==================================================
               number of      length   percentage
               elements*    occupied  of sequence
--------------------------------------------------
SINEs:            17794      1061170 bp    0.09 %
      ALUs          363        31340 bp    0.00 %
      MIRs         1166        92129 bp    0.01 %

LINEs:             4456       888114 bp    0.08 %
      LINE1         976       103929 bp    0.01 %
      LINE2         813        82891 bp    0.01 %
      L3/CR1        699        63627 bp    0.01 %

LTR elements:      1187       199118 bp    0.02 %
      ERVL          155        15828 bp    0.00 %
      ERVL-MaLRs    200        20737 bp    0.00 %
      ERV_classI    379        42833 bp    0.00 %
      ERV_classII    66         6896 bp    0.00 %

DNA elements:      2290       196866 bp    0.02 %
     hAT-Charlie    190        15468 bp    0.00 %
     TcMar-Tigger   732        37473 bp    0.00 %

Unclassified:       101        12946 bp    0.00 %

Total interspersed repeats:  2358214 bp    0.20 %


Small RNA:         5954       433422 bp    0.04 %

Satellites:         366        55705 bp    0.00 %
Simple repeats:  310641     14322152 bp    1.22 %
Low complexity:   47381      2844279 bp    0.24 %
==================================================

* most repeats fragmented by insertions or deletions
  have been counted as one element
  Runs of >=20 X/Ns in query were excluded in % calcs


The query species was assumed to be homo sapiens  
RepeatMasker Combined Database: Dfam_Consensus-20170127, RepBase-20170127
        
run with rmblastn version 2.6.0+

RUN 2 (species – Crassostrea gigas)

Output folder:

Summary table (text):

Output table (GFF):

SUMMARY TABLE

==================================================
file name: jelly.out.fasta          
sequences:        696946
total length: 1253001795 bp  (1172226648 bp excl N/X-runs)
GC level:         36.51 %
bases masked:  160759267 bp ( 13.71 %)
==================================================
               number of      length   percentage
               elements*    occupied  of sequence
--------------------------------------------------
Retroelements       213132     69887654 bp    5.96 %
   SINEs:             2374       311974 bp    0.03 %
   Penelope         171792     57862186 bp    4.94 %
   LINEs:           195605     63430615 bp    5.41 %
    CRE/SLACS            0            0 bp    0.00 %
     L2/CR1/Rex        731       357995 bp    0.03 %
     R1/LOA/Jockey       0            0 bp    0.00 %
     R2/R4/NeSL         13        11377 bp    0.00 %
     RTE/Bov-B        8085      1948581 bp    0.17 %
     L1/CIN4             0            0 bp    0.00 %
   LTR elements:     15153      6145065 bp    0.52 %
     BEL/Pao          2119       955773 bp    0.08 %
     Ty1/Copia         101        75372 bp    0.01 %
     Gypsy/DIRS1     11776      4815361 bp    0.41 %
       Retroviral        0            0 bp    0.00 %

DNA transposons     256292     35689117 bp    3.04 %
   hobo-Activator    19847      2059651 bp    0.18 %
   Tc1-IS630-Pogo    43269      6806311 bp    0.58 %
   En-Spm                0            0 bp    0.00 %
   MuDR-IS905            0            0 bp    0.00 %
   PiggyBac           7935      1060296 bp    0.09 %
   Tourist/Harbinger  9503       887332 bp    0.08 %
   Other (Mirage,        0            0 bp    0.00 %
    P-element, Transib)

Rolling-circles          0            0 bp    0.00 %

Unclassified:       174943     38299211 bp    3.27 %

Total interspersed repeats:   143875982 bp   12.27 %


Small RNA:             280        78768 bp    0.01 %

Satellites:           7383      1362194 bp    0.12 %
Simple repeats:     278809     12982714 bp    1.11 %
Low complexity:      44078      2622506 bp    0.22 %
==================================================

* most repeats fragmented by insertions or deletions
  have been counted as one element
  Runs of >=20 X/Ns in query were excluded in % calcs


The query species was assumed to be crassostrea gigas
RepeatMasker Combined Database: Dfam_Consensus-20170127, RepBase-20170127
        
run with rmblastn version 2.6.0+

RUN 3 (species – Crassostrea virginica)

Output folder:

Summary table (text):

Output table (GFF):

SUMMARY TABLE

==================================================
file name: jelly.out.fasta          
sequences:        696946
total length: 1253001795 bp  (1172226648 bp excl N/X-runs)
GC level:         36.51 %
bases masked:   39598953 bp ( 3.38 %)
==================================================
               number of      length   percentage
               elements*    occupied  of sequence
--------------------------------------------------
Retroelements        63882     10327611 bp    0.88 %
   SINEs:            63882     10327611 bp    0.88 %
   Penelope              0            0 bp    0.00 %
   LINEs:                0            0 bp    0.00 %
    CRE/SLACS            0            0 bp    0.00 %
     L2/CR1/Rex          0            0 bp    0.00 %
     R1/LOA/Jockey       0            0 bp    0.00 %
     R2/R4/NeSL          0            0 bp    0.00 %
     RTE/Bov-B           0            0 bp    0.00 %
     L1/CIN4             0            0 bp    0.00 %
   LTR elements:         0            0 bp    0.00 %
     BEL/Pao             0            0 bp    0.00 %
     Ty1/Copia           0            0 bp    0.00 %
     Gypsy/DIRS1         0            0 bp    0.00 %
       Retroviral        0            0 bp    0.00 %

DNA transposons       9433      2307292 bp    0.20 %
   hobo-Activator        0            0 bp    0.00 %
   Tc1-IS630-Pogo        0            0 bp    0.00 %
   En-Spm                0            0 bp    0.00 %
   MuDR-IS905            0            0 bp    0.00 %
   PiggyBac              0            0 bp    0.00 %
   Tourist/Harbinger     0            0 bp    0.00 %
   Other (Mirage,        0            0 bp    0.00 %
    P-element, Transib)

Rolling-circles          0            0 bp    0.00 %

Unclassified:        51558      9836468 bp    0.84 %

Total interspersed repeats:    22471371 bp    1.92 %


Small RNA:           64164     10406776 bp    0.89 %

Satellites:             10         5985 bp    0.00 %
Simple repeats:     298612     14185090 bp    1.21 %
Low complexity:      47510      2866522 bp    0.24 %
==================================================

* most repeats fragmented by insertions or deletions
  have been counted as one element
  Runs of >=20 X/Ns in query were excluded in % calcs


The query species was assumed to be crassostrea virginica
RepeatMasker Combined Database: Dfam_Consensus-20170127, RepBase-20170127
        
run with rmblastn version 2.6.0+

RUN 4 (species – Ostrea lurida)

Output folder:

Summary table (text):

Output table (GFF):

SUMMARY TABLE

==================================================
file name: jelly.out.fasta          
sequences:        696946
total length: 1253001795 bp  (1172226648 bp excl N/X-runs)
GC level:         36.51 %
bases masked:   17617763 bp ( 1.50 %)
==================================================
               number of      length   percentage
               elements*    occupied  of sequence
--------------------------------------------------
Retroelements            0            0 bp    0.00 %
   SINEs:                0            0 bp    0.00 %
   Penelope              0            0 bp    0.00 %
   LINEs:                0            0 bp    0.00 %
    CRE/SLACS            0            0 bp    0.00 %
     L2/CR1/Rex          0            0 bp    0.00 %
     R1/LOA/Jockey       0            0 bp    0.00 %
     R2/R4/NeSL          0            0 bp    0.00 %
     RTE/Bov-B           0            0 bp    0.00 %
     L1/CIN4             0            0 bp    0.00 %
   LTR elements:         0            0 bp    0.00 %
     BEL/Pao             0            0 bp    0.00 %
     Ty1/Copia           0            0 bp    0.00 %
     Gypsy/DIRS1         0            0 bp    0.00 %
       Retroviral        0            0 bp    0.00 %

DNA transposons          0            0 bp    0.00 %
   hobo-Activator        0            0 bp    0.00 %
   Tc1-IS630-Pogo        0            0 bp    0.00 %
   En-Spm                0            0 bp    0.00 %
   MuDR-IS905            0            0 bp    0.00 %
   PiggyBac              0            0 bp    0.00 %
   Tourist/Harbinger     0            0 bp    0.00 %
   Other (Mirage,        0            0 bp    0.00 %
    P-element, Transib)

Rolling-circles          0            0 bp    0.00 %

Unclassified:            3          189 bp    0.00 %

Total interspersed repeats:         189 bp    0.00 %


Small RNA:             282        79165 bp    0.01 %

Satellites:             10         5985 bp    0.00 %
Simple repeats:     313082     14662647 bp    1.25 %
Low complexity:      47785      2878201 bp    0.25 %
==================================================

* most repeats fragmented by insertions or deletions
  have been counted as one element
  Runs of >=20 X/Ns in query were excluded in % calcs


The query species was assumed to be ostrea lurida 
RepeatMasker Combined Database: Dfam_Consensus-20170127, RepBase-20170127
        
run with rmblastn version 2.6.0+

BS-seq Mapping – Olympia oyster bisulfite sequencing: TrimGalore > FastQC > Bismark

Steven asked me to evaluate our methylation sequencing data sets for Olympia oyster.

According to our Olympia oyster genome wiki, we have the following two sets of BS-seq data:

All computing was conducted on our Apple Xserve: roadrunner.

All steps were documented in this Jupyter Notebook (GitHub): 20180503_emu_oly_methylation_mapping.ipynb

NOTE: The Jupyter Notebook linked above is very large in size. As such it will not render on GitHub. It will need to be downloaded to a computer that can run Jupyter Notebooks and viewed that way.

Here’s a brief overview of what was done.

Samples were trimmed with TrimGalore and then evaluated with FastQC. MultiQC was used to generate a nice visual summary report of all samples.

The Olympia oyster genome assembly, pbjelly_sjw_01, was used as the reference genome and was prepared for use in Bismark:


/home/shared/Bismark-0.19.1/bismark_genome_preparation \
--path_to_bowtie /home/shared/bowtie2-2.3.4.1-linux-x86_64/ \
--verbose /home/sam/data/oly_methylseq/oly_genome/ \
2> 20180507_bismark_genome_prep.err

Bismark was run on trimmed samples with the following command:


/home/shared/Bismark-0.19.1/bismark \
--path_to_bowtie /home/shared/bowtie2-2.3.4.1-linux-x86_64/ \
--genome /home/sam/data/oly_methylseq/oly_genome/ \
-u 1000000 \
-p 16 \
--non_directional \
/home/sam/analyses/20180503_oly_methylseq_trimgalore/1_ATCACG_L001_R1_001_trimmed.fq.gz \
/home/sam/analyses/20180503_oly_methylseq_trimgalore/2_CGATGT_L001_R1_001_trimmed.fq.gz \
/home/sam/analyses/20180503_oly_methylseq_trimgalore/3_TTAGGC_L001_R1_001_trimmed.fq.gz \
/home/sam/analyses/20180503_oly_methylseq_trimgalore/4_TGACCA_L001_R1_001_trimmed.fq.gz \
/home/sam/analyses/20180503_oly_methylseq_trimgalore/5_ACAGTG_L001_R1_001_trimmed.fq.gz \
/home/sam/analyses/20180503_oly_methylseq_trimgalore/6_GCCAAT_L001_R1_001_trimmed.fq.gz \
/home/sam/analyses/20180503_oly_methylseq_trimgalore/7_CAGATC_L001_R1_001_trimmed.fq.gz \
/home/sam/analyses/20180503_oly_methylseq_trimgalore/8_ACTTGA_L001_R1_001_trimmed.fq.gz \
/home/sam/analyses/20180503_oly_methylseq_trimgalore/zr1394_10_s456_trimmed.fq.gz \
/home/sam/analyses/20180503_oly_methylseq_trimgalore/zr1394_11_s456_trimmed.fq.gz \
/home/sam/analyses/20180503_oly_methylseq_trimgalore/zr1394_12_s456_trimmed.fq.gz \
/home/sam/analyses/20180503_oly_methylseq_trimgalore/zr1394_13_s456_trimmed.fq.gz \
/home/sam/analyses/20180503_oly_methylseq_trimgalore/zr1394_14_s456_trimmed.fq.gz \
/home/sam/analyses/20180503_oly_methylseq_trimgalore/zr1394_15_s456_trimmed.fq.gz \
/home/sam/analyses/20180503_oly_methylseq_trimgalore/zr1394_16_s456_trimmed.fq.gz \
/home/sam/analyses/20180503_oly_methylseq_trimgalore/zr1394_17_s456_trimmed.fq.gz \
/home/sam/analyses/20180503_oly_methylseq_trimgalore/zr1394_18_s456_trimmed.fq.gz \
/home/sam/analyses/20180503_oly_methylseq_trimgalore/zr1394_1_s456_trimmed.fq.gz \
/home/sam/analyses/20180503_oly_methylseq_trimgalore/zr1394_2_s456_trimmed.fq.gz \
/home/sam/analyses/20180503_oly_methylseq_trimgalore/zr1394_3_s456_trimmed.fq.gz \
/home/sam/analyses/20180503_oly_methylseq_trimgalore/zr1394_4_s456_trimmed.fq.gz \
/home/sam/analyses/20180503_oly_methylseq_trimgalore/zr1394_5_s456_trimmed.fq.gz \
/home/sam/analyses/20180503_oly_methylseq_trimgalore/zr1394_6_s456_trimmed.fq.gz \
/home/sam/analyses/20180503_oly_methylseq_trimgalore/zr1394_7_s456_trimmed.fq.gz \
/home/sam/analyses/20180503_oly_methylseq_trimgalore/zr1394_8_s456_trimmed.fq.gz \
/home/sam/analyses/20180503_oly_methylseq_trimgalore/zr1394_9_s456_trimmed.fq.gz \
2> 20180507_bismark_02.err

Results:

TrimGalore output folder:

FastQC output folder:

MultiQC output folder:

MultiQC Report (HTML):

Bismark genome folder: 20180503_oly_genome_pbjelly_sjw_01_bismark/

Bismark output folder:


Whole genome BS-seq (2015)

Prep overview
  • Library prep: Roberts Lab
  • Sequencing: Genewiz
Bismark Report Mapping Percentage
1_ATCACG_L001_R1_001_trimmed_bismark_bt2_SE_report.txt 40.3%
2_CGATGT_L001_R1_001_trimmed_bismark_bt2_SE_report.txt 39.9%
3_TTAGGC_L001_R1_001_trimmed_bismark_bt2_SE_report.txt 40.2%
4_TGACCA_L001_R1_001_trimmed_bismark_bt2_SE_report.txt 40.4%
5_ACAGTG_L001_R1_001_trimmed_bismark_bt2_SE_report.txt 39.9%
6_GCCAAT_L001_R1_001_trimmed_bismark_bt2_SE_report.txt 39.6%
7_CAGATC_L001_R1_001_trimmed_bismark_bt2_SE_report.txt 39.9%
8_ACTTGA_L001_R1_001_trimmed_bismark_bt2_SE_report.txt 39.7%

MBD BS-seq (2015)

Prep overview
  • MBD: Roberts Lab
  • Library prep: ZymoResearch
  • Sequencing: ZymoResearch
Bismark Report Mapping Percentage
zr1394_1_s456_trimmed_bismark_bt2_SE_report.txt 33.0%
zr1394_2_s456_trimmed_bismark_bt2_SE_report.txt 34.1%
zr1394_3_s456_trimmed_bismark_bt2_SE_report.txt 32.5%
zr1394_4_s456_trimmed_bismark_bt2_SE_report.txt 32.8%
zr1394_5_s456_trimmed_bismark_bt2_SE_report.txt 35.2%
zr1394_6_s456_trimmed_bismark_bt2_SE_report.txt 35.5%
zr1394_7_s456_trimmed_bismark_bt2_SE_report.txt 32.8%
zr1394_8_s456_trimmed_bismark_bt2_SE_report.txt 33.0%
zr1394_9_s456_trimmed_bismark_bt2_SE_report.txt 34.7%
zr1394_10_s456_trimmed_bismark_bt2_SE_report.txt 34.9%
zr1394_11_s456_trimmed_bismark_bt2_SE_report.txt 30.5%
zr1394_12_s456_trimmed_bismark_bt2_SE_report.txt 35.8%
zr1394_13_s456_trimmed_bismark_bt2_SE_report.txt 32.5%
zr1394_14_s456_trimmed_bismark_bt2_SE_report.txt 30.8%
zr1394_15_s456_trimmed_bismark_bt2_SE_report.txt 31.3%
zr1394_16_s456_trimmed_bismark_bt2_SE_report.txt 30.7%
zr1394_17_s456_trimmed_bismark_bt2_SE_report.txt 32.4%
zr1394_18_s456_trimmed_bismark_bt2_SE_report.txt 34.9%

Assembly Comparisons – Oly Assemblies Using Quast

I ran Quast to compare all of our current Olympia oyster genome assemblies.

See Jupyter Notebook in Results section for Quast execution.

Results:

Output folder: http://owl.fish.washington.edu/Athaliana/quast_results/results_2018_01_16_10_08_35/

Heatmapped table of results: http://owl.fish.washington.edu/Athaliana/quast_results/results_2018_01_16_10_08_35/report.html

Very enlightening!

After all the difficulties with PB Jelly, it has produced the most large contigs. However, it does also have the highest quantity and rate of N’s of all the assemblies produced to date.

BEST OF:

# contigs (>= 50000 bp): pbjelly_sjw_01 (894)
Largest Contig: redundans_sjw_02 (322,397bp)
Total Length: pbjelly_sjw_01 (1,180,563,613bp)
Total Length (>=50,000bp): pbjelly_sjw_01 (57,741,906bp)
N50: redundans_sjw_03 (17,679bp)

Jupyter Notebook (GitHub): 20180116_swoose_oly_assembly_comparisons_quast.ipynb

Genome Assembly – Olympia Oyster Illumina & PacBio Using PB Jelly w/BGI Scaffold Assembly

After another attempt to fix PB Jelly, I ran it again.

We’ll see how it goes this time…

Re-ran this using the BGI Illumina scaffolds FASTA.

Here’s a brief rundown of how this was run:

See the Jupyter Notebook for full details of run (see Results section below).

Results:

Output folder: http://owl.fish.washington.edu/Athaliana/20171130_oly_pbjelly/

Output FASTA file: http://owl.fish.washington.edu/Athaliana/20171130_oly_pbjelly/jelly.out.fasta

Quast assessment of output FASTA:

Assembly jelly.out
# contigs (>= 0 bp) 696946
# contigs (>= 1000 bp) 159429
# contigs (>= 5000 bp) 68750
# contigs (>= 10000 bp) 35320
# contigs (>= 25000 bp) 7048
# contigs (>= 50000 bp) 894
Total length (>= 0 bp) 1253001795
Total length (>= 1000 bp) 1140787867
Total length (>= 5000 bp) 932263178
Total length (>= 10000 bp) 691523275
Total length (>= 25000 bp) 261425921
Total length (>= 50000 bp) 57741906
# contigs 213264
Largest contig 194507
Total length 1180563613
GC (%) 36.57
N50 12433
N75 5983
L50 26241
L75 60202
# N’s per 100 kbp 6580.58

Have added this assembly to our Olympia oyster genome assemblies table.

This took an insanely long time to complete (nearly six weeks)!!! After some internet searching, I’ve found a pontential solution to this and have initiated another PB Jelly run to see if it will run faster. Regardless, it’ll be interesting to see how the results compare from two independent runs of PB Jelly.

Jupyter Notebook (GitHub): 20171130_emu_pbjelly.ipynb

Assembly Comparison – Oly Assemblies Using Quast

I ran Quast to compare all of our current Olympia oyster genome assemblies.

See Jupyter Notebook in Results section for Quast execution.

Results:

Output folder: http://owl.fish.washington.edu/Athaliana/quast_results/results_2017_11_14_12_30_25/

Heatmapped table of results: http://owl.fish.washington.edu/Athaliana/quast_results/results_2017_11_14_12_30_25/report.html

Very enlightening!

BEST OF:

Largest Contig: redundans_sjw_02 (322,397bp)
Total Length: soap_bgi_01 & pbjelly_sjw_01 (697,528,655bp)
Total Length (>=50,000bp): redundans_sjw_03 (17,006,058bp)
N50: redundans_sjw_03 (17,679bp)

Interesting tidbit: The pbjelly_sjw_01 assembly is EXACTLY the same as the soap_bgi_01. Looking at the output messages from that PB Jelly assembly, one can see why. The messages indicate that no gaps were filled on the BGI scaffold reference! That means the PB Jelly output is just the BGI scaffold reference assembly!

Jupyter Notebook (GitHub): 20171114_swoose_oly_assembly_comparisons_quast.ipynb

Genome Assembly – Olympia Oyster Illumina & PacBio Using PB Jelly w/BGI Scaffold Assembly

Yesterday, I ran PB Jelly using Sean’s Platanus assembly, but that didn’t produce an assembly because PB Jelly was expecting gaps in the Illumina reference assembly (i.e. scaffolds, not contigs).

Re-ran this using the BGI Illumina scaffolds FASTA.

Here’s a brief rundown of how this was run:

See the Jupyter Notebook for full details of run (see Results section below).

Results:

Output folder: http://owl.fish.washington.edu/Athaliana/20171114_oly_pbjelly/

Output FASTA file: http://owl.fish.washington.edu/Athaliana/20171114_oly_pbjelly/jelly.out.fasta

OK! This seems to have worked (and it was quick, like less than an hour!), as it actually produced a FASTA file! Will run QUAST with this and some assemblies to compare assembly stats. Have added this assembly to our Olympia oyster genome assemblies table.

Jupyter Notebook (GitHub): 20171114_emu_pbjelly_BGI_scaffold.ipynb

Genome Assembly – Olympia Oyster Illumina & PacBio Using PB Jelly w/Platanus Assembly

Sean had previously attempted to run PB Jelly, but ran into some issues running on Hyak, so I decided to try this on Emu.

Here’s a brief rundown of how this was run:

See the Jupyter Notebook for full details of run (see Results section below).

Results:

Output folder: http://owl.fish.washington.edu/Athaliana/20171113_oly_pbjelly/

This completed very quickly (like, just a couple of hours). I also didn’t experience the woes of multimillion temp file production that killed Sean’s attempt at running this on Mox (Hyak).

However, it doesn’t seem to have produced an assembly!

Looking through the output, it seems as though it didn’t produce an assembly because there weren’t any gaps to fill in the reference. This makes sense (in regards to the lack of gaps in the reference Illumina assembly) because I used the Platanus contig FASTA file (i.e. not a scaffolds file). I didn’t realize PB Jelly was just designed for gap filling. Guess I’ll give this another go using the BGI scaffold FASTA file and see what we get.

Jupyter Notebook (GitHub): 20171113_emu_pbjelly_22mer_plat.ipynb

Software Crash – Olympia oyster genome assembly with Masurca on Mox

Ah, the joys of bioinformatics. I just received an email from Mox indicating that the Masurca assembly I started 11 DAYS AGO (!!) crashed.

I’m probably not going to put much effort in to trying to figure out what went wrong, but here’s some log file snippets for reference. I’ll probably drop a line to the developers and see if they have any easy ways to address whatever caused the problems, but that’s about as much effort as I’m willing to put into troubleshooting this assembly.

Additionally, since this crashed, I’m not going to bother moving any of the files off of Mox. That means they will be deleted automatically by the system around Nov. 9th, 2017.


slurm-94620.out (tail)

compute_psa 6601202 2632582819
Refining alignments
Joining
Generating assembly input files
Coverage of the mega-reads less than 5 -- using the super reads as well
Coverage threshold for splitting unitigs is 138 minimum ovl 63
Running assembly
/gscratch/srlab/programs/MaSuRCA-3.2.3/bin/deduplicate_unitigs.sh: line 85: 24330 Aborted                 (core dumped) overlapStoreBuild -o $ASM_DIR/$ASM_PREFIX.ovlStore -M 65536 -g $ASM_DIR/$ASM_PREFIX.gkpStore $ASM_DIR/overlaps_dedup.ovb.gz > $ASM_DIR/overlapStore.rebuild.err 2>&1
Assembly stopped or failed, see CA.mr.41.15.17.0.029.log
[Mon Oct 30 23:19:37 PDT 2017] Assembly stopped or failed, see CA.mr.41.15.17.0.029.log

CA.mr.41.15.17.0.029.log (tail)

number of threads     = 28 (OpenMP default)

ERROR:  overlapStore '/gscratch/scrubbed/samwhite/20171019_masurca_oly_assembly/CA.mr.41.15.17.0.029/genome.ovlStore' is incomplete; previous overlapStoreBuild probably crashed.

----------------------------------------
Failure message:

failed to unitig

overlapStore.rebuild.err

Scanning overlap files to count the number of overlaps.
Found 277.972 million overlaps.
Memory limit 65536MB supplied.  Ill put 3246167525 IIDs (3435.97 million overlaps) into each of 1 buckets.
bucketizing CA.mr.41.15.17.0.029/overlaps_dedup.ovb.gz
bucketizing DONE!
overlaps skipped:
               0 OBT - low quality
               0 DUP - non-duplicate overlap
               0 DUP - different library
               0 DUP - dedup not requested
terminate called after throwing an instance of std::bad_alloc
  what():  std::bad_alloc

Failed with Aborted

Backtrace (mangled):

overlapStoreBuild[0x40523a]
/usr/lib64/libpthread.so.0(+0xf100)[0x2af83b3c0100]
/usr/lib64/libc.so.6(gsignal+0x37)[0x2af83c0395f7]
/usr/lib64/libc.so.6(abort+0x148)[0x2af83c03ace8]
/usr/lib64/libstdc++.so.6(_ZN9__gnu_cxx27__verbose_terminate_handlerEv+0x165)[0x2af83b62d9d5]
/usr/lib64/libstdc++.so.6(+0x5e946)[0x2af83b62b946]
/usr/lib64/libstdc++.so.6(+0x5e973)[0x2af83b62b973]
/usr/lib64/libstdc++.so.6(+0x5eb93)[0x2af83b62bb93]
/usr/lib64/libstdc++.so.6(_Znwm+0x7d)[0x2af83b62c12d]
/usr/lib64/libstdc++.so.6(_Znam+0x9)[0x2af83b62c1c9]
overlapStoreBuild[0x402e10]
/usr/lib64/libc.so.6(__libc_start_main+0xf5)[0x2af83c025b15]
overlapStoreBuild[0x403089]

Backtrace (demangled):

[0] overlapStoreBuild() [0x40523a]
[1] /usr/lib64/libpthread.so.0::(null) + 0xf100  [0x2af83b3c0100]
[2] /usr/lib64/libc.so.6::(null) + 0x37  [0x2af83c0395f7]
[3] /usr/lib64/libc.so.6::(null) + 0x148  [0x2af83c03ace8]
[4] /usr/lib64/libstdc++.so.6::__gnu_cxx::__verbose_terminate_handler() + 0x165  [0x2af83b62d9d5]
[5] /usr/lib64/libstdc++.so.6::(null) + 0x5e946  [0x2af83b62b946]
[6] /usr/lib64/libstdc++.so.6::(null) + 0x5e973  [0x2af83b62b973]
[7] /usr/lib64/libstdc++.so.6::(null) + 0x5eb93  [0x2af83b62bb93]
[8] /usr/lib64/libstdc++.so.6::operator new(unsigned long) + 0x7d  [0x2af83b62c12d]
[9] /usr/lib64/libstdc++.so.6::operator new[](unsigned long) + 0x9  [0x2af83b62c1c9]
[10] overlapStoreBuild() [0x402e10]
[11] /usr/lib64/libc.so.6::(null) + 0xf5  [0x2af83c025b15]
[12] overlapStoreBuild() [0x403089]

GDB:

Genome Assembly – Olympia oyster Illumina & PacBio Reads Using Redundans

Had problems with Docker and Jupyter Notebook inexplicably dying and deleting all the files in the working directory of the Jupyter Notebook (which also happened to be the volume mounted in the Docker container).

So, I ran this on my computer, but didn’t have Jupyter installed (yet).

This utilized the Canu contigs file (FASTA) that I generated on 20171018.

Here’s the input command:

sudo python /home/sam/software/redundans/redundans.py -t 24 -l m130619_081336_42134_c100525122550000001823081109281326_s1_p0.fastq.gz m170211_224036_42134_c101073082550000001823236402101737_s1_X0_filtered_subreads.fastq.gz m170301_100013_42134_c101174162550000001823269408211761_s1_p0_filtered_subreads.fastq.gz m170301_162825_42134_c101174162550000001823269408211762_s1_p0_filtered_subreads.fastq.gz m170301_225711_42134_c101174162550000001823269408211763_s1_p0_filtered_subreads.fastq.gz m170308_163922_42134_c101174252550000001823269408211742_s1_p0_filtered_subreads.fastq.gz m170308_230815_42134_c101174252550000001823269408211743_s1_p0_filtered_subreads.fastq.gz m170315_001112_42134_c101169372550000001823273008151717_s1_p0_filtered_subreads.fastq.gz m170315_063041_42134_c101169382550000001823273008151700_s1_p0_filtered_subreads.fastq.gz m170315_124938_42134_c101169382550000001823273008151701_s1_p0_filtered_subreads.fastq.gz m170315_190851_42134_c101169382550000001823273008151702_s1_p0_filtered_subreads.fastq.gz -i 151114_I191_FCH3Y35BCXX_L1_wHAIPI023992-37_1.fq.gz 151114_I191_FCH3Y35BCXX_L1_wHAIPI023992-37_2.fq.gz 151114_I191_FCH3Y35BCXX_L2_wHAMPI023991-66_1.fq.gz 151114_I191_FCH3Y35BCXX_L2_wHAMPI023991-66_2.fq.gz 151118_I137_FCH3KNJBBXX_L5_wHAXPI023905-96_1.fq.gz 151118_I137_FCH3KNJBBXX_L5_wHAXPI023905-96_2.fq.gz 160103_I137_FCH3V5YBBXX_L3_WHOSTibkDCABDLAAPEI-62_1.fq.gz 160103_I137_FCH3V5YBBXX_L3_WHOSTibkDCABDLAAPEI-62_2.fq.gz 160103_I137_FCH3V5YBBXX_L3_WHOSTibkDCACDTAAPEI-75_1.fq.gz 160103_I137_FCH3V5YBBXX_L3_WHOSTibkDCACDTAAPEI-75_2.fq.gz 160103_I137_FCH3V5YBBXX_L4_WHOSTibkDCABDLAAPEI-62_1.fq.gz 160103_I137_FCH3V5YBBXX_L4_WHOSTibkDCABDLAAPEI-62_2.fq.gz 160103_I137_FCH3V5YBBXX_L4_WHOSTibkDCACDTAAPEI-75_1.fq.gz 160103_I137_FCH3V5YBBXX_L4_WHOSTibkDCACDTAAPEI-75_2.fq.gz 160103_I137_FCH3V5YBBXX_L5_WHOSTibkDCAADWAAPEI-74_1.fq.gz 160103_I137_FCH3V5YBBXX_L5_WHOSTibkDCAADWAAPEI-74_2.fq.gz 160103_I137_FCH3V5YBBXX_L6_WHOSTibkDCAADWAAPEI-74_1.fq.gz 160103_I137_FCH3V5YBBXX_L6_WHOSTibkDCAADWAAPEI-74_2.fq.gz -f 20171018_oly_pacbio.contigs.fasta -o /home/data/20171024_docker_oly_redundans_01/

This completed in just over 19hrs.

Copied output files to Owl: http://owl.fish.washington.edu/Athaliana/20171024_docker_oly_redundans_01/

Here’s the desired output file (FASTA): scaffolds.reduced.fa

Will add to our genome assemblies table.

Ran Quast on 20171103 for some assembly stats.

Quast output is here: http://owl.fish.washington.edu/Athaliana/quast_results/results_2017_11_03_22_43_06/