Lab notebook of Steven Roberts
Today we sampled Jesse, the Olympia oyster selected for genome sequencing. Not as big of a specimen as yesterday, but made up for it with video.
The oyster was collected in Fidalgo Bay, weighing in at 58.99 grams and 70 mm long.
The adductor muscle was selected for genome sequencing, thus quickly rinsed in 10% bleach.
Other tissues were sampled for RNA-seq
Samples were placed in two boxes, for redundancy.
Today we sampled the geoduck (Panopea generosa) for genome sequencing. Here is how things went down.
It was an early morning for the clam, peaking out to see a glorious sunrise on the porch.
From there it was off to the lab.
After cleaning the surfaces, Brent sampled tissue.
We started out started out targetting the foot and adductor muscles. These tissues were steriley removed and then rinsed in 1% bleach, followed by Nanopure water. This tissue will be used for genome sequencing as we predict least amount of associated taxa.
Remaining tissues were taken, primarily for RNA-seq and divided into two boxes.
Tubes were labeled on cap with tissue type.
Here is what Box 1 looks like.
Box 2 looks the same however it does not have a heart or style sample.
The only surpise was in sampling, labial palps were identified after we had already sampled a pair.
Taking the most decent Ct values, I did the simple and crude calculation, normalizing with EF1 and looking at fold over minimum.
Seems to be EF1 data is skewing. Will take a look with actin and also compare delta Ct, using this as a sound reference.
One of the premises related to germline methylation in oysters is that genes with limited methylation will have more alternatively spliced products. One of our most robust identification of alternatively spliced genes is from the heat shock experiment, however methylation was assessed with array to ID DMRs. In order to gauge the relationship of germline methylation and alternative splicing I looked at all of our data and made the calls.
I simply looked at the 42 areas determined to be alternatively spliced in the three individuals and qualitatively made the call. For example..
Of the 42 genes, only 11 were determined have limited methylation.
scaffold1391:350297-393525 - High scaffold1501:189-2280 - Low-Medium scaffold1546:22946-41272 - High scaffold157:93056-102166 - High scaffold157:288396-298950 - High scaffold1583:636568-663717 - High scaffold1630:57333-61806 - Low scaffold1643:190932-200778 - Low scaffold1670:360106-365501 - High scaffold1750:71251-77856 - High scaffold1009:677703-719650 - High scaffold1009:990592-1008075 - High scaffold193:111771-117728 - High scaffold198:1032767-1055090 - Medium-High scaffold198:1084454-1102022 - High scaffold211:954418-990421 - Medium-High scaffold351:641567-648889 - Low scaffold102:1297353-1322657 - Low-High scaffold383:99975-117650 - Low-High scaffold38366:25577-54928 - Low scaffold1024:1037898-1043721 - Medium-High scaffold395:85069-105025 - High scaffold399:120007-128313 - High scaffold41228:55316-64219 - Low scaffold41858:126164-135361 - High scaffold42366:124115-157800- High scaffold42892:55315-57225 - Low scaffold42904:154963-177485 - High scaffold43208:19552-46201 - High scaffold43940:65971-85144 - High scaffold452:65648-77493 - Low scaffold527:16020-64639 - Low scaffold588:178668-183438 - High scaffold616:53220-63262 - Low scaffold828:110697-114691 - High scaffold942:369690-377892 - High scaffold1160:333463-390372 - Low-High scaffold1213:118721-121110 - Low scaffold128:428337-438047 - Low scaffold1282:23471-48121 - Low scaffold13:4222-16204 - Low scaffold1322:265134-304245 - High
The caveat is (there always seems to be one) is this list of alternative spliced genes is based on consistency across individuals, something we would predict NOT to see if this was truly a stochastic phenomenon.
Today Sam and I took care of the daily maintenance of F2 Olympia oysters grown at the Ken Chew Shellfish Hatchery at Manchester, WA. In short, larvae are being described from Fidalgo (North: NF), Oyster Bay (South: SS), and Hood Canal (HC) broodstock. Though I had done most aspects before, never without Katherine, thus she provided instructions.
When I arrived the algae tank was empty and pump was off.
The system was flushed with freshwater (with Sam soaked with said water) along with bleach. The algae tank was filled. The first set of tanks that were processed were the larger larvae (+160um).
Using 100um screen larvae were collected, brought up in 800ml and 0.5ml (x3) taken for counts.
The remaining larvae were placed back into cleaned systems.
Next, the smaller larvae tanks were processed, using 160 over 100 um screens, with larger ones moving over to +160um tanks (above). For this counts were done for both size classes and DNA samples taken for 160 size class. The smaller larvae placed back in the same tanks.
For DNA samples, 4mls of larvae from 800ml beaker were taken, rinsed with ethanol and placed in 1.5ml centrifuge tube with 1ml of RNAlater (This was also done with fresh larvae- below).
The last systems tackled were the “new”, fresh larvae from collectors..
For this, larvae were captured using 200um / 100um screen, with larvae moved to larger tanks, counts done, and samples collected for DNA. As expected very little larvae, they were brought up in 400ml, 1ml used for counting, and 8ml used for DNA samples.
Sam counted the larvae, probably still very wet (Sam and the larvae).
And here are the counts!
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
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
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
Here is first look at annotations from a gonad transcriptome assembly.
Also available @ https://raw.githubusercontent.com/sr320/paper-Geoduck-proteomic/master/analyses/Pgen_uniprot_ID-2.csv
For purposes of proposaling and reports, I have gone back to look at a small project done in collaboration with scientist at IFREMER looking at pesticide exposure on oyster larvae methylation.
The control library had limited yield so the number of loci with data from the treated and the control library was restricted. However using a liberal 3x coverage for both, we found a total of 823 DMRs (544 hypermethylated and 279 hypomethylated).
Intriguingly, when one accounts for all CGs in the genome, these DMRs are predominantly in transposable elements.
Reminiscent of …
The analysis is not pretty, but here is what I have to offer.
https://github.com/sr320/nb-2015/blob/master/Cg/YE-pesticide/YE-pesticide-summary.ipynb
Here is a summary of 10 Dramatically Differentially Methylated Regions (DDMRs), that is, those covered in this post that are found across a majority of the oysters exposed to acute heat shock.
—
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].”
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.
Significant hypomethylation is also present within the intron of Tenascin, a glycoprotein expressed in the extracellular matrix during stress.
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.
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.
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.
Another interesting DMR was found in Methylated-DNA–protein-cysteine methyltransferase.
Within the intron of a Nacrein-like protein is a hypomethylated DMR. This is a negative regulator of calcification in shells of mollusks.
Collagen alpha-1(IV) chain is another gene that contains a hypomethylated DMR. This protein is the major structural component of basement membranes.
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.
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