Tag Archives: updated

Finishing out with the mechanical

Currently there is a pretty robust spreadsheet and over the past few days Jake has cranked through some reps to see how the oysters that were mechanically stressed hold up. Below is how these data are integrated.

Jake_Heare_Research_Central__8_10_2015_EF1d_Mech_Stress_2_rep_qPCR_1B821B44.png

Currently the 8-10 samples (yellow) have been skipped, but we might have a look.

First up is having a look at the new HSP 70 reps. The mechanical data still needs some better resolution. Hopefully teh 8-10 samples migh shed some light.

Screenshot_8_17_15__9_53_AM_1B821ECC.png

Next up is two more reps of PGEEP4.
Looks good, and given the doubling of reps we could easily drop ‘outlier’ runs and still have triplicates, tight triplicates.

Screenshot_8_17_15__10_15_AM_1B822404.png

GRB2… now good to go, with the first pair of reps dead on.

BMP2…. could use some help from the other mechanical stress runs
Screenshot_8_17_15__11_03_AM_1B822F48.png

TLR….seemed like a relatively easy fix (besides no detection) in that just needed to correct for machine.
Screenshot_8_17_15__11_10_AM_1B8230E7.png

And the correction indicating the fact that expression was so low, only able to be detected by Opticon
Screenshot_8_17_15__11_17_AM_1B82327D.png

The 8-15 runs had minimal control and temp samples with mechanical run in dups.

Screenshot_8_17_15__11_24_AM_1B82344E.png

This needs a little carressing before integrating into data.
This should be in two columns with empty cells where no samples were run- in this order.

H_C_1
H_C_2
H_C_3
H_C_4
H_C_5
H_C_6
H_C_7
H_C_8
N_C_1
N_C_2
N_C_3
N_C_4
N_C_5
N_C_6
N_C_7
N_C_8
S_C_1
S_C_2
S_C_3
S_C_4
S_C_5
S_C_6
S_C_7
S_C_8
H_T_1
H_T_2
H_T_3
H_T_4
H_T_5
H_T_6
H_T_7
H_T_8
N_T_1
N_T_2
N_T_3
N_T_4
N_T_5
N_T_6
N_T_7
N_T_8
S_T_1
S_T_2
S_T_3
S_T_4
S_T_5
S_T_6
S_T_7
S_T_8
H_M_1
H_M_2
H_M_3
H_M_4
H_M_5
H_M_6
H_M_7
H_M_8
N_M_1
N_M_2
N_M_3
N_M_4
N_M_5
N_M_6
N_M_7
N_M_8
S_M_1
S_M_2
S_M_3
S_M_4
S_M_5
S_M_6
S_M_7
S_M_8

8-15 run update

Actin
Screenshot_8_17_15__3_55_PM_1B8273B6.png

Mechanical looks decent after correcting.

However taken together, bothersome the difference in crude expression levels.
Screenshot_8_17_15__3_57_PM_1B827441.png

Carm

Had some wet works issues

Jake_Heare_Research_Central__8_15_2015_CARM_CTM2_reps_qPCR_1B8274C8.png

H2AV
Assuming correction is correct- still a big differences in mechanincal here- could be real.

Screenshot_8_17_15__4_11_PM_1B8277A0.png

PGRP
No correction required as these were run on cfx, downside is some reps are not detected that would have been picked up with Opticon.

Do not see be shift in expression of mechanical stressed.
Screenshot_8_17_15__4_18_PM_1B827900.png

CRAF
Easy correction but skeptical of some very, very low Cts

Screenshot_8_17_15__4_25_PM_1B827AA2.png

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Second look at Geoduck transcriptome

Last week I popped out a quick assembly and annotation on our geoduck gonadal transcriptome. A second assembly was also done using Trinity.


Updates
August 3 – Confirmed // in file location had no impact on assembly.
July 14 – TransDecoder protein annotations
10:40am – added TransDecoder results
10:29am – added Stats via Trinity


Trinity.pl 
--seqType fq 
-JM 24G 
--left /Volumes/web/cnidarian/Geo_Pool_F_GGCTAC_L006_R1_001_val_1.fq /Volumes/web/cnidarian/Geo_Pool_M_CTTGTA_L006_R1_001_val_1.fq 
--right /Volumes/web/cnidarian//Geo_Pool_F_GGCTAC_L006_R2_001_val_2.fq /Volumes/web/cnidarian//Geo_Pool_M_CTTGTA_L006_R2_001_val_2.fq 
--CPU 16 

trinity_out_dir_1B54203C.png

Output

0:999   127840
1000:1999   18164
2000:2999   5321
3000:3999   1817
4000:4999   762
5000:5999   291
6000:6999   135
7000:7999   73
8000:8999   22
9000:9999   29
10000:10999     4
11000:11999     5
12000:12999     3
13000:13999     4
14000:14999     4
15000:15999     3
16000:16999     0
17000:17999     2
18000:18999     1

Total length of sequence:   101862868 bp
Total number of sequences:  154480
N25 stats:          25% of total sequence length is contained in the 8095 sequences >= 2045 bp
N50 stats:          50% of total sequence length is contained in the 26158 sequences >= 1014 bp
N75 stats:          75% of total sequence length is contained in the 64574 sequences >= 446 bp
Total GC count:         37657770 bp
GC %:               36.97 %
hummingbird:Geo-trinity steven$ /Users/gilesg/compile/trinityrnaseq_r20131110/util/TrinityStats.pl /Volumes/web/cnidarian/Geo-trinity/trinity_out_dir/Trinity.fasta 


################################
## Counts of transcripts, etc.
################################
Total trinity transcripts:  154480
Total trinity components:   100155
Percent GC: 36.97

########################################
Stats based on ALL transcript contigs:
########################################

    Contig N10: 3444
    Contig N20: 2385
    Contig N30: 1766
    Contig N40: 1343
    Contig N50: 1014

    Median contig length: 371
    Average contig: 659.39
    Total assembled bases: 101862868


#####################################################
## Stats based on ONLY LONGEST ISOFORM per COMPONENT:
#####################################################

    Contig N10: 2999
    Contig N20: 2026
    Contig N30: 1462
    Contig N40: 1067
    Contig N50: 768

    Median contig length: 321
    Average contig: 553.88
    Total assembled bases: 55473621

Rerunning to see if double slash was a problem- did not see anything in error. Also running TransDecoder


TransDecoder Results

Ran the following

/Users/gilesg/compile/trinityrnaseq_r20131110/trinity-plugins/TransDecoder_r20131110/TransDecoder -t  /Volumes/web/cnidarian/Geo-trinity/trinity_out_dir/Trinity.fasta

This provided a peptide file with 36003 sequences.

!head /Volumes/web-1/cnidarian/Geo-trinity/Trinity.fasta.transdecoder.pep

>cds.comp100047_c0_seq2|m.5982 comp100047_c0_seq2|g.5982 ORF comp100047_c0_seq2|g.5982 comp100047_c0_seq2|m.5982 type:internal len:142 (-) comp100047_c0_seq2:3-425(-)
NAECRDLYKIFTQILSVRSQEGKIVIPDEFATKIRNWLGNKEELFKEAHNQKIITFYNEY
TREENTFNPIRGKRPMSVPDMPERKYIDQLSRKTQSQCDFCKYKTFTAEDTFGRIDSNFS
CSASNAFKLDHWHALFLLKTH


Running blastp on Trinity.fasta.transdecoder.pep

!blastp 
-query /Volumes/web/cnidarian/Geo-trinity/Trinity.fasta.transdecoder.pep 
-db /usr/local/bioinformatics/dbs/uniprot_sprot.fasta 
-evalue 1e-5 
-max_target_seqs 1 
-max_hsps 1 
-outfmt 6 
-num_threads 4 
-out /Volumes/web/cnidarian/Geo-trinity/Trinity.fasta.transdecoder.pep-blastp-uniprot-2.out

results: http://eagle.fish.washington.edu/cnidarian/Geo-trinity/Trinity.fasta.transdecoder.pep-blastp-uniprot-2.out

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A closer look at DMRs

A detailed look at DMRs that hold true across oysters exposed to heat shock.


Updated July 6, 2015 – added three more DMRs (at bottom of post)


Going down the list, scaffold418_576986 is a feature that overlaps gene EKC36328, Bromodomain-containing protein 8. Specifically the location is in the intron between exon 18 and 19 (total of 20 exons). This gene is differentially expressed, that is expressed at an elevated level following heat shock.

“The precise function of the domain is unclear, but it may be involved in protein-protein interactions and may play a role in assembly or activity of multi-component complexes involved in transcriptional activation [PMID: 7580139].”

scaffold418_576986


Another DMR that is consistent across oysters is located within the intron of Homeobox protein LOX2. Homeobox are transcription factors often associated with developmental processes.

scaffold247_141885


Significant hypomethylation is also present within the intron of Tenascin, a glycoprotein expressed in the extracellular matrix during stress.

scaffold1518_212680


We also found a DMR upstream of E3 ubiquitin-protein ligase UHRF1. Interestingly this is a protein that bridges DNA methylation and chromatin modification.

“Specifically recognizes and binds hemimethylated DNA at replication forks via its YDG domain and recruits DNMT1 methyltransferase to ensure faithful propagation of the DNA methylation patterns through DNA replication. In addition to its role in maintenance of DNA methylation, also plays a key role in chromatin modification: through its tudor-like regions and PHD-type zinc fingers, specifically recognizes and binds histone H3 trimethylated at ‘Lys-9′ (H3K9me3) and unmethylated at ‘Arg-2′ (H3R2me0), respectively, and recruits chromatin proteins. Enriched in pericentric heterochromatin where it recruits different chromatin modifiers required for this chromatin replication. Also localizes to euchromatic regions where it negatively regulates transcription possibly by impacting DNA methylation and histone modifications. Has E3 ubiquitin-protein ligase activity by mediating the ubiquitination of target proteins such as histone H3 and PML. It is still unclear how E3 ubiquitin-protein ligase activity is related to its role in chromatin in vivo.” ~http://www.uniprot.org/uniprot/Q96T88

While not classified as a differentially expressed gene, there does appear to be a trend towards increased expression upon heat stress. This occurrence would follow the traditional model where decreased methylation in the promoter region is associated with increased expression.

scaffoldscaffold853_46186


There were three features identified that are in fact within an intron of Methylcrotonoyl-CoA carboxylase subunit alpha, mitochondrial, and enzyme involved in leucine and isovaleric acid catabolism.

scaffold406


In an another example of a DMR associated with a differentially expressed gene, a DMR that span an intron and exon within Myosin heavy chain, striated muscle. In this case the gene is expressed at a lower level upon heat stress. It is also worth pointing out this gene has very limited methylation overall based on other studies we have done.

scaffold394_555813


Another interesting DMR was found in Methylated-DNA–protein-cysteine methyltransferase.

scaffold242_75918


Within the intron of a Nacrein-like protein is a hypomethylated DMR. This is a negative regulator of calcification in shells of mollusks.

scaffold142_656144


Collagen alpha-1(IV) chain is another gene that contains a hypomethylated DMR. This protein is the major structural component of basement membranes.

scaffold12_243960


The only DMR that is hypermethylated is odd in the fact that annotation was dropped one the data was integrated into Ensembl. This could be related to the fact that the closest blast hit to this gene model is Insertion element IS1 protein insA and transposable element in prokaryotes.

scaffold257_1235165

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First steps at an aggregated view of all DNA methylation data (updated)

Seems like I have gotten close (see here) but do not have a canonical IGV session that has all of our DNA methylation data. The goal here is to generate such a product (and publish, so I do not lose it).

All data is publicly available at

http://owl.fish.washington.edu/halfshell/index.php?dir=2015-05-comgenbro

see also data on Figshare


Updates

July 2, 2015 – added Heat Shock experiment alternative splice track
June 26, 2015 – add link to Figshare version
June 26, 2015 – updated Archive.zip
June 26, 2015 – added numerous array tracks from heat stress array experiment including 3+ tracks.
June 26, 2015 – added new track from heat stress – Heat-multi-individual-dmr.bed
June 22, 2015 – updated Archive.zip
June 22, 2015 – updated MBD-seq track gills (no bisulfite treatment) to use unique mapping (see also [this](MBD-seq track gills (no bisulfite treatment))
June 22, 2015 – Updated EE2 linkout to go to Github
June 22, 2015 – Corrected error in labelling EE2 experiment tracks
June 15, 2015 – added MBD-seq track gills (no bisulfite treatment)
June 15, 2015 – added larval pesticide treatment tracks (bisulfite treatment)
June 15, 2015 – new IGV screenshot
June 15, 2015 – added HS-Cuffdiff_geneexp.sig.gtf (differentially expressed genes from heat-shock)

 

 


Metadata

FileID Description Links
Crassostrea_gigas.GCA_000297895.1.26.gtf gtf ftp
MBD-Gill-meth MBD enriched DNA library alignment paper, info
BiGill_CpG_methylation gill methylation 5x (MBD-BS, hi output) paper
BiGill_exon_clc_rpkm Corresponding exon-specific gene expression paper
BiGo_CpG_methylation male gamete methylation 5x (hi output) paper
M1 male gamete methylation 5x preprint
M3 male gamete methylation 5x preprint
T1D3 72hpf larvae from M1 methylation 5x preprint
T1D5 120hpf larvae from M1 methylation 5x preprint
T3D3 72hpf larvae from M3 methylation 5x preprint
T3D5 120hpf larvae from M3 methylation 5x preprint
Heat-multi-individual-dmr.bed Heat Stress (13 locations) common signal notebook
2M_3plusmerge_Hyper.bed merging adj probes to single interval notebook
2M_3plusmerge_Hypo.bed merging adj probes to single interval notebook
4M_3plusmerge_Hyper.bed merging adj probes to single interval notebook
4M_3plusmerge_Hypo.bed merging adj probes to single interval notebook
6M_3plusmerge_Hyper.bed merging adj probes to single interval notebook
6M_3plusmerge_Hypo.bed merging adj probes to single interval notebook
2M_Hyper_3plusAdjactentProbes.gff 3+ adjacent probes notebook
2M_Hypo_3plusAdjactentProbes.gff 3+ adjacent probes notebook
4M_Hyper_3plusAdjactentProbes.gff 3+ adjacent probes notebook
4M_Hypo_3plusAdjactentProbes.gff 3+ adjacent probes notebook
6M_Hyper_3plusAdjactentProbes.gff 3+ adjacent probes notebook
6M_Hypo_3plusAdjactentProbes.gff 3+ adjacent probes notebook
2M_sig Heat stress DMRs (array), ind.#2 notebook, draft
4M_sig Heat stress DMRs (array), ind.#4 notebook, draft
6M_sig Heat stress DMRs (array), ind.#6 notebook, draft
HS-Cuffdiff_geneexp.sig.gtf Heat stress differentially expressed genes notebook
HS-Cuffdiff_altsplice.bed Heat stress alternatively spliced genes notebook
2M.bedgraph.tdf RNA-seq from ind.#2 above – pretreament notebook, draft
4M.bedgraph.tdf RNA-seq from ind.#4 above – pretreament notebook, draft
6M.bedgraph.tdf RNA-seq from ind.#6 above – pretreament notebook, draft
2M-HS.bedgraph.tdf RNA-seq from ind.#2 above – post-heatshock notebook, draft
4M-HS.bedgraph.tdf RNA-seq from ind.#4 above – post-heatshock notebook, draft
6M-HS.bedgraph.tdf RNA-seq from ind.#6 above – post-heatshock notebook, draft
mgaveryDMRs_112212.gff EE2 exposure DMRs (array) paper
A01.smoothed EE2 exposure array data – input versus input paper
A02.smoothed EE2 exposure array data – EE2 vs control paper
A03.smoothed EE2 exposure array data – EE2 vs control (dyeswap) paper
YE_mixHYPER.bed DMRs in pesticide exposed larvae (hypermethylated)
YE_mixHYPO.bed DMRs in pesticide exposed larvae (hypomethylated)
YE_mix_22smCG3x larvae (mix pesticide exposed) methylation
YE_control_22smCG3x larvae (control) methylation

screenshot

anyone should be able to render this in IGV with this session file:

http://owl.fish.washington.edu/halfshell/2015-05-comgenbro/igv_session.xml


This work was supported in part by the National Science Foundation (NSF) under Grant Number 1158119 awarded to SR Roberts

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