- Miscellaneous 658
- Geoduck Genome Sequencing 71
- Olympia oyster reciprocal transplant 67
- Olympia Oyster Genome Sequencing 64
- PROPS 36
- Computer Servicing 24
- Crassostrea gigas larvae OA (2011) bisulfite sequencing 24
- Samples Submitted 23
- LSU C.virginica Oil Spill MBD BS Sequencing 22
- 2bRAD Library Tests for Sequencing at Genewiz 22
- Genotype-by-sequencing at BGI 22
- Goals 20
- Protein expression profiles during sexual maturation in Geoduck 14
- Samples Received 13
- Lineage-specific DNA methylation patterns in developing oysters 11
- BS-seq Libraries for Sequencing at Genewiz 11
- Sea star RNA-seq 10
- MBD Enrichment for Sequencing at ZymoResearch 8
- Reagent Prep 7
- SRA Submissions 5
- Myostatin Interacting Proteins 3
- Tanner Crab RNAseq 3
- Olypia Oyster Genome Sequencing 1
After a meeting on this project yesterda, we decided to try a few things to continue with various approaches to assessing the metagenome. One of the approaches is to run BLASTx on the individual water sample MEGAHIT assemblies from 20190327 and obtain taxonomy info for them, so that’s what I did here.
Earlier today, we received the additional G.gigas sequencing data from Genewiz. Wanted to run through FastQC again and get an updated report for each data set. Admittedly, it probably won’t look much different from the initial FastQC run on 20190415, due to the fact that the additional sequencing was simply appended to the previous data. Since FastQC examines a subset of the data in each file, I’d fully expect the FastQC report to look the same. However, we’ll have a greater number of sequences in each file. This should, in turn, increase the number of reads retained after quality trimming.
The FastQC analysis of the intitial data we received from Genewiz (on 20190408)showed consistent failures in the “Per Tile Sequence Quality” for all of Roberto’s Crassostrea gigas sequencing. After discussing with Genewiz, they offered to generate an additional 25% reads for each of those libraries.
After the success we had isolating RNA using the Quick-DNA/RNA Microprep Plus Kit (ZymoResearch), Steven had me isolate RNA from a list of ~117 samples. Of that list, I was able to find 66 crab hemolymph pelleted RNAlater samples. The “missing” samples were most likely previosly used by Grace during our various attempts to get some usable RNA out these.
A while ago, Steven tasked me with assessing some questions related to the sequencing coverage we get doing MBD-BSseq in this GitHub issue. At the heart of it all was really to try to get an idea of how much usable data we actually get when we do an MBD-BSseq project. Yaamini happened to have done an MBD-BSseq project relatively recently, and it’s one she’s actively working on analyzing, so we went with that data set.
Nearing the end of this quick metagenomics comparison of taxonomic differences between the two pH treatments (pH=7.1 and pH=8.2). Previously ran:
Continuing with a relatively quick comparison of pH treatments (pH=7.1 vs. pH=8.2), I wanted to run gene prediction on the MEGAHIT assemblies I made yesterday. I ran MetaGeneMark on the two pH-specific assemblies on Mox. This should be a very fast process (I’m talking, like a couple of minutes fast), so it enhances the annotation with very little effort and time.
A report involving our work on the geoduck water metagenomics is due later this week and our in-depth analysis for this project using Anvi’o is likely to require at least another week to complete. Even though we have a broad overview of the metagenomic taxa present in these water samples, we don’t have data in a format for comparing across samples/treatments. So, I initiated our simplified pipeline (MEGAHIT > MetaGeneMark > BLASTn > KronaTools) for examining our metagenomic data of the two treatments:
In the continuing struggle to isolate RNA from the Chionoecetes bairdi hemolymph preserved in RNAlater, Pam managed to find the Quick-DNA-RNA Microprep Plus Kit (ZymoResearch) as a potential option. We received a free sample of the kit and so I gave it a shot. Grace pulled 10 samples she’d previously used to isolate RNA (and was unable to get anything out of virtually all of them using the RNeasy Plus Micro Kit (Qiagen)) for me to try out this new kit:
I previously assembled and annotated P.generosa larval Day 5 transcriptome (20190318 - mislabeled as Juvenile Day 5 in my previous notebook entries) using just our HiSeq data from our Illumina collaboration. This was a an oversight, as I didn’t realize that we also had NovaSeq RNAseq data. So, I’ve initiated another de novo assembly using Trinity incorporating both sets of data.
I previously assembled and annotated P.generosa gonad transcriptome (20190318) using just our HiSeq data from our Illumina collaboration. This was a an oversight, as I didn’t realize that we also had NovaSeq RNAseq data. So, I’ve initiated another de novo assembly using Trinity incorporating both sets of data.
I previously assembled and annotated P.generosa ctenidia transcriptome (20190318) using just our HiSeq data from our Illumina collaboration. This was a an oversight, as I didn’t realize that we also had NovaSeq RNAseq data. So, I’ve initiated another de novo assembly using Trinity incorporating both sets of data.
Received the WGBS data from Genewiz that were sent to Genewiz for whole genome bisulfite sequencing on 20190207. These were from:
Continuing work on the metagenomics project, Emma shared her “co-assembly”, so I figured it would be quick and easy to compare hers with mine and get a feel for how different/similar they might be. I did a similar comparison last week where I compared each of our individual water sample assemblies. Those results showed my assemblies generated:
As part of addressing this GitHub Issue on assessing taxonomic diversity in our metagenomics samples, I decided to compare the individual sample assemblies I made on 20190327 using Megahit and the assemblies that Emma made. Emma utilized NGless on her cluster in Genome Sciences with the following scripts:
I decided to give Anvi’o a shot for this metagenomics analysis, as it seems ridiculously thorough and easy to use, with a nice-looking, interactive plotting interface. Additionally, they have a TON of clearly written tutorials on how to use their software and explanations of what’s happening when you use it! Really, couldn’t ask for much more. So, here’s how it went…
Crassostrea virginica samples that were subjected to MBD enrichment (completed 20190319) were shipped, on dry ice, to ZymoResearch today for BSseq. They will perform bisulfite conversion, library prep, and sequencing. Sequencing output is to be ~50M paired-end reads (at least 100bp or 151bp) per library.
In preparation for some manuscripts, Steven asked that I get some geoduck RNAseq data upload to the NCBI sequencing read archive (SRA) in this GitHub Issue. Specifically, he needed the data corresponding to day 5 (pos-fertilization) larve RNAseq data that was prepped/run by Illumina on the NovaSeq. Original sample submission notebook entry is here.
Continuing on getting the metagenomics sequencing project written up as a manuscript, Steven asked me to provide an overview of the taxonomic makeup of our metagenome assembly in this GitHub Issue. Earlier today, I ran analysis using BLASTp data. I initiated additional analysis using the MetaGeneMark nucleotide data to run BLASTn on Mox.
Finished MBD enrichment on 20190312 using the MethylMiner kit. Since we were out of Qubit assay tubes, I could not quantify these samples when I initially completed the ethanol precipitation. Tubes are in, so I went forward with quantification using the Roberts’ Lab Qubit 3.0 and the 1x High Sensitivity dsDNA assay.
Last week I performed MBD enrichment on 12 of the 24 Lotterhos C.viriginica mantle DNA on samples I had sheared on 20190306. I proceeded with MBD enrichment using the MethylMiner Kit (Invitrogen) on the 12 remaining samples of the 24. I followed the manufacturer’s protocol for input DNA >1ug - 10ug with the following modifications:
Yesterday, I sheared the Lotterhos C.virginica gDNA in preparation for MBD enrichment. Today, I proceeded with MBD enrichment using the MethylMiner Kit (Invitrogen) on 12 of the 24 samples. I followed the manufacturer’s protocol for input DNA >1ug - 10ug with the following modifications:
A little while ago, we installed some additional hard drives in Gannet (Synology RS3618XS) with the intention of expanding the total storage space. However, the original set of HDDs were set up as RAID10. As it turns out, RAID10 configurations cannot be expanded! So, the new set of HDDs were configured as a separate volume (Volume 2) in a RAID6 configuration. After backing up the
/volume1/web directory (via
rsync) to our UW Google Drive, I begane the data migration.
Ran BUSCO on our completed annotation of the P.generosa v071 genome (GFF) (subset of sequences >10kbp). See this notebook entry for genome annotation info. This provides a nice metric on how “complete” a genome assembly (or transcriptome) is. Additionally, BUSCO is tied in with Augustus for gene prediction and generates ab initio gene models. With that said, since I just want to evaluate the completeness of this particular genome assembly, I’ll be using the annotated genome generated through two rounds of SNAP gene prediction. Otherwise, I’d use the initial MAKER annotations to generate an Augustus gene model that could be used in conjuction with the SNAP models (I’ll likely do this at a later date).
Steven tasked me with processing ~90 FastA files containing gene sequences from C.virginica in this GitHub Issue. He needed to determine the Observed/Expected (O/E) ratio of CpGs in each FastA. He provided this example code and this link to all the files. Additionally, today, he tasked Kaitlyn with merging all of the output CpG O/E values for each sample in to a single file, but I decided to tackle it anyway.
With the P.generosa v070 annotation taking a very long time (due to the genome/transcriptome sizes, and, hitting some snags in the script I put together), Steven asked if I could subset the genome and annotate the v071 assembly (>10kbp subset) in order to have some annotations to use for some talks coming up ASLO (in this GitHub issue).
Last week, I isolated DNA from all of Ronti’s ctenidia samples, however one sample (D18-C) didn’t isolate properly. So, I performed another isolation procedure with another section of frozen tissue. Tissue was excised from frozen tissue block via razor blade (weight not recorded) and pulverized under liquid nitrogen. Samples were incubated O/N @ 37oC (heating block) in 350uL of MB1 Buffer + 25uL Proteinase K, per the E.Z.N.A. Mollusc DNA Kit (Omega) instructions.
Using the C.gigas cytochrome c oxidase (COX1) primers (SR IDs: 1713, 1714)I designed the other day, I ran a qPCR on a subset of Ronit’s diploid/triploid control/heat shocked oyster DNA that Shelly had previously isolated and performed global DNA methylation assay. The goal is to get a rough assessment of whether or not there appear to be differences in relative mitochondrial abundances between these samples.
Apparently we’ve had some interest in the Pacific herring transcriptomes (liver and testes) we produced many years ago. As such, Steven asked that I do a quick BLASTx to help annotate these two transcriptomes.
Steven tasked me with assembling our geoduck metagenomics HiSeqX data. The first part of the process is examining the quality of the sequencing reads, performing quality trimming, and then checking the quality of the trimmed reads. It’s also possible (likely) that I’ll need to run another round of trimming. The process is documented in the Jupyter Notebook linked below. After these reads are cleaned up, I’ll transfer them over to our HPC nodes (Mox) and try assembling them.
We’re attempting a quick & dirty comparison of relative mitochondria counts between diploid and triploid C.gigas, so needed a single-copy mitochondrial gene target for qPCR. Selected cytochrome c oxidase subunit 1 (COX1), based on a quick glance at the NCBI mt genome browser for C.gigas NC_001276.
Earlier today I made some cDNA from geoduck gonad RNA for use in this qPCR to test out the vitellogenin primers I designed on 20181129
In preparation for designing primers for developing a geoduck vitellogenin qPCR assay, I annotated a de novo geoduck transcriptome assembly last week. Next up, identify vitellogenin genes, design primers, confirm their specificity, and order them!
I was tasked with generating some qPCR primers to analyze vitellogenin expression in geoduck. In order to do so, I needed to annotate a geoduck transcriptome in order to identify potential vitellogenin genes. I had previously assembled a geoduck transcriptome. For annotation, I used TransDecoder (v5.5.0). The annotation was run on our Mox HPC node.
After confirming Ronit’s DNased RNA was free of gDNA, I quantified the DNased RNA from 20181115 using the Roberts Lab Qubit 3.0 and the Qubit hsRNA Assay.
Isolated DNA from the Lotterhos Crassostrea virginica mantle samples received on 20181017 using the E.Z.N.A. Mollusc Kit, with the following modifications/notes:
Proceeded with reverse transcription of [Ronit’s DNased ctenidia RNA (from 20181016)(2018-10-16-dnase-treatment-ronits-c-gigas-ploiyddessication-ctenidia-rna.html).
After I figured out the appropriate DNA and primers to use to detect gDNA in Crassostrea gigas samples, I checked Ronit’s [DNased ctenidia RNA (from 20181016)(2018-10-16-dnase-treatment-ronits-c-gigas-ploiyddessication-ctenidia-rna.html) for residual gDNA.
The qPCR I ran earlier today to check for residual gDNA in Ronit’s DNased RNA turned out terribly, due to a combination of bad primers and, possibly, bad gDNA.
I previously isolated RNA from crab hemolymp from a lyophilized sample using TriReagent and Grace recently tried isolating RNA from crab hemolyph pellet (non-lyophilized) using TriReagent. The results for her extractions weren’t so great, so I’m giving it a shot with the following samples:
Steven recently saw an announcement that Microsoft R Open now handles multi-threaded processing (default R does not), so we were interested in trying it out. I installed MLR/MRO on Emu/Roadrunner (Apple Xserve; Ubuntu 16.04). Followed the Microsoft installation directions for Ubuntu. In retrospect, I think I could’ve just installed MRO, but this gets the job done as well and won’t hurt anything.
Used all of our current oly RNAseq data to assemble a transcriptome using Trinity.
I previously ran two variations on the Bismark analysis for our Olympia oyster whole genome bisulfite sequencing data:
Due to difficulties getting RNA from hemolymph samples stored in RNAlater, Grace is testing out lyophilizing samples before extraction. Who knows what impact this will have on RNA, but it’s worth a shot!
Ran Bismark using our high performance computing (HPC) node, Mox, with two different bowtie2 settings:
We previously received sea lice (Caligus tape) DNA from Cris Gallardo-Escarate at Universidad de Concepción.
NOTE: IMPORTANT CAVEATS - READ POST BEFORE USING DATA.
Used all of our current geoduck RNAseq data to assemble a transcriptome using Trinity.
Bismark analysis of all of our current Olympia oyster (Ostrea lurida) DNA methylation high-throughput sequencing data.
Isolated RNA from 40 Tanner crab hemolymph samples selected by Grace with the RNeasy Plus Micro Kit (Qiagen) according to the manufacturer’s protocol, with the following modifications:
In a continued attempt to figure out what we can do about the tanner crab RNA, Steven tasked me with using an RNeasy Kit to cleanup some existing RNA.
The high performance computing (HPC) cluster (called Mox) at Univ. of Washington (UW) frustratingly requires a password when SSH-ing, even when SSH keys are in use. I have a lengthy, unintelligible password that I use for my UW account, so having to type this in any time I want to initiate a new SSH session on Mox is a painful process.
I previously performed this analysis using a different version of our Ostrea lurida genome assembly. [Steven asked that I repeat the analysis with a modified version of the genome assembly (Olurida_v081)(https://github.com/RobertsLab/resources/issues/265#issuecomment-401055771) - only has contigs >1000bp in length.
Made Illumina libraries with goeduck metagenome water filter DNA I previously isolated on:
After yesterday’s difficulties getting RMblast to compile, I deleted the folder and went through the build process again.
Steven asked that I re-run some Olympia oyster transposable elements analysis using RepeatMasker and a newer version of our Olympia oyster genome assembly.
Earlier this week, I ran TrimGalore!, but set the trimming, incorrectly - due to a copy/paste mistake, as
--non-directional, so I re-ran with the correct settings.
I ran a subset of Yaamini’s ocean chemistry samples on our T5 Excellence titrator (Mettler Toledo) at the beginning of April. The subset were samples taken from the beginning, middle, and end of the experiment. The rationale for this was to assess whether or not total alkalinity (TA) varied across the experiment. If there was little variation, then there’d likely be no need to run all of the samples. However, should there be temporal differences, then all samples should be processed.
Steven found out that the Bismarck documentation (Bismarck is the bisulfite aligner we use in our BS-seq pipeline) suggests trimming 10bp from both the 5’ and 3’ ends. Since this is the next step in our pipeline, we figured we should probably just follow their recommendations!
Isolated DNA from the following two filters:
Earlier today, I ran TrimGalore/FastQC/MultiQC on the Crassostrea virginica MBD BS-seq data from ZymoResearch and hard trimmed the first 14bp from each read. Things looked better at the 5’ end, but the 3’ end of each of the READ1 seqs showed a wonky 2bp blip, so decided to trim that off.
Yesterday, I ran TrimGalore/FastQC/MultiQC on the Crassostrea virginica MBD BS-seq data from ZymoResearch with the default settings (i.e. “auto-trim”). There was still some variability in the first ~15bp of the reads and Steven wanted to see how a hard trim would change things.
Yesterday, I ran FastQC/MultiQC on the Crassostrea virginica MBD BS-seq data from ZymoResearch. Steven wanted to trim it and see how things turned out.
This is another attempt to isolate DNA from two more of the geoduck hatchery metagenome samples Emma delivered on 20180313.
Isolated DNA from two of the geoduck hatchery metagenome samples Emma delivered on 20180313 to get an idea of what type of yields we might get from these.
Performed total alkalinity (TA) titrations on Hollie’s samples using our T5 Excellence titrator (Mettler Toledo) and Rondolino sample changer.
Performed total alkalinity (TA) titrations on Hollie’s samples using our T5 Excellence titrator (Mettler Toledo) and Rondolino sample changer.
I’ll begin this entry with a TL;DR (becuase it’s definitely a very long read):
I’ve been working on getting our T5 Excellence titrator (Mettler Toledo) with Rondolino sample changer (Mettler Toledo) set up and operational.
The two MspI restriction digestions from earlier today for our project with Qiagen were subjected to phenol:chloroform cleanup and subsequent ethanol precipitations.
I figured it’d be prudent to collect some Eastern oyster (Crassotrea virginica) to have around the lab.
After I finally resolved the installation of PB Jelly on Emu (running Ubuntu 16.04), I’ve had a PB Jelly assembly running for the past two weeks! I’ve gotten tired of checking on its status (i.e. is it still running?) every day, so I dove in and figured out how to set up Emu to email me when the job is complete!
DNA was isolated from a single adult Eastern oyster (Crassostrea virginica) for a pilot project with Qiagen to test their new DNA bisulfite conversion kit. The oyster was obtained yesterday afternoon (20171210) from the Taylo rShellfish Pioneer Square location. The oyster was stored @ 4C O/N.
I previously installed and ran PB Jelly. Despite no error messages being output, I noticed something odd during my quick post-assembly stats check: The PB Jelly numbers were identical to the input reference file. This seemed very strange and made me decide to look a bit deeper in the PB Jelly output files.
I isolated DNA from the Crassotrea virginica gonad samples sent by Katie Lotterhos using the E.Z.N.A. Mollusc Kit with the following modifications:
List of software that needed installing to run ALPACA:
Ah, the joys of bioinformatics. I just received an email from Mox indicating that [the Masurca assembly I started 11 DAYS AGO (!!)(2017/10/19/genome-assembly-olympia-oyster-illumina-pacbio-reads-using-masurca.html) crashed.
I recently finished an assembly of our Olympia oyster PacBio data using Canu and thought it would be interesting to compare to Sean’s Canu assembly.
Saw this tweet this morning and thought this would be good to try out for our Olympia oyster genome assemblies, as it will handle “hybrid” assemblies (i.e. short-reads and long reads):
Uh, not sure what happened here:
My previous go at this was a little premature - I didn’t wait for Laura to fully annotate her slides/blocks. Little did I know, the tissue was mostly visceral mass and, as such, I didn’t hit much in the way of actual gonad tissue. So, I’m redoing this, now that Grace has gone through and annotated the blocks to point out gonad tissue. SN-10-16 was sent to Katherine Silliman on 20170720.
UPDATE 20170712: The RNA I isolated below is from incorrect regions of tissue. I misunderstood exactly what this tissue was, and admittedly, jumped the gun. The tissue is actually collected from the visceral mass - which contains gonad (a small amount) and digestive gland (a large amount). The RNA isolated below will be stored in one of the Shellfish RNA boxes and I will isolate RNA from the correct regions indicated by Grace
I’ve been asked to isolate RNA from some paraffin-embedded Olympia oyster gonad tissue.
Updated a couple of GitHub Wikis:
Used the MethylFlash Methylated DNA Quantification Kit (Colorimetric) from Epigentek to quantify methylation in these coral DNA samples.
Three samples (of the 62 total) that were quantified earlier today, had concentrations too low for use in the methylation assay:
I previously highlighted some of the issues I was having using Authorea.com as an writing platform.
Jay received notice from UC Berkeley that the sequencing data from his coral RADseq was ready. In addition, the sequencing contains some epiRADseq data from samples provided by Hollie Putnam. See his notebook for multiple links that describe library preparation (indexing and barcodes), sample pooling, and species breakdown.
I’m currently trying to write a manuscript covering our genotype-by-sequencing data for the Olympia oyster using the Authorea.com platform and am encountering some issues that are a bit frustrating. Here’s what’s happening (and the ways I’ve managed to get around the problems).
Yesterday, I downloaded the Illumina FASTQ files provided by Genewiz for Hollie Putnam’s reduced representation bisulfite geoduck libraries. However, Genewiz had not provided a checksum file at the time.
Hollie Putnam prepared some reduced representation bisulfite Illumina libraries and had them sequenced by Genewiz.
Last week I downloaded the final BGI data files and assemblies for Olympia oyster and geoduck genome sequencing projects. However, the output from the download command made the Jupyter Notebook files too large to view and/or upload to GitHub. So, I had to trim the output cells from that notebook in order to render it usable/viewable.
Previously downloaded Jay’s epiRADseq data that was provided by the Genomic Sequencing Laboratory at UC-Berkeley. It was provided already demultiplexed (which is very nice of them!). To be completionists on our end, we requested the non-demultiplexed data set.
I helped Katherine Silliman with her oyster sampling today from her ocean acidification experiment with Olympia oysters (Ostrea lurida) at the Kenneth K. Chew Center for Shellfish Research & Restoration, which is housed at the NOAA Northwest Fisheries Science Center at Manchester in a partnership with the [Puget Sound Restoration Fund (PSRF)(http://www.restorationfund.org/). We sampled the following tissues and stored in 1mL RNAlater:
Submitted the following manuscript to PeerJ for peer review:
I recently moved some computing jobs over to Amazon’s Elastic Cloud Computing (EC2) in attempt to avoid some odd computing issues/errors I kept encountering on our lab computers (Apple Xserve 3,1).
Well, I tackled the storage space issue by expanding the EC2 Instance to have a 1000GB of storage space. Now that that’s no longer a concern, it turns out I’m running up against processing/memory limits!
We’re working on a project with Washington Department of Natural Resources’ (DNR) Micah Horwith to identify potential proteomic biomarkers in geoduck (Panopea generosa) and Pacific oyster (Crassostrea gigas). One aspect of the project is how to best conduct sampling of juvenile geoduck (Panopea generosa) and Pacific oyster (Crassostrea gigas) to minimize changes in the proteome of ctenidia tissue during sampling. Generally, live animals are shucked, tissue dissected, and then the tissue is “snap” frozen. However, Micah’s crew will be collecting animals from wild sites around Puget Sound and, because of the remote locations and the means of collection, will have limited tools and time to perform this type of sampling. Time is a significant component that will have great impact on proteomic status in each individual.
This all takes a surprisingly long time to set up.
One liner to create Docker container for Jupyter notebook usage and data analysis on roadrunner (Xserve):
This isn’t really a notebook entry - it’s more of a traditional blog post.
I decided to run a quick test to see what difference in speed (i.e. time) we might see between handling files that are stored locally, on an external hard drive (HDD), or on our server (Owl).
We have an enormous backlog of high-throughput sequencing files (641 FASTQ files, to be exact) that we need/want to get added to the NCBI Sequence Read Archive (SRA).
We’ve had a recent influx of sequencing data, which is great, but it created a bit of a backlog documenting what we’ve received.
Ran a PCR to obtain luciferase DNA for sequencing.
Jake and Steven recently noticed localized “hot spots” on most of Jake’s recent qPCRs, where higher levels of fluorescence were consistently showing up in interior portions of the plates than the outer portion of the plates.
Performed reverse transcription on the Olympia oyster DNased RNA from the control samples and the 1hr heat shock samples of Jake’s project. To accommodate the large numbers of anticipated genes to be targeted in subsequent qPCRs, I prepared 100μL reactions (normally, 25μL reactions are prepared) using 250ng of each DNased RNA. A 1:10 dilution of the oligo dT primers (Promega) was prepared to improve pipetting accuracy. All incubations were performed in a thermal cycler without using a heated lid.
Prepared two DNA plates and corresponding primer plates for sequencing at the UW HTGC from the purified gel-purified PCRs from yesterday. Primer plates were prepared by adding 7μL of NanoPure H2O to each well and then adding 3μL of 10μM primer to the appropriate wells. For the DNA plates, added 10μL of DNA to the appropriate wells.
Purified DNA from the remaining PCR bands excised by Jake on 20150609 and 20150610, as well as Jonathan’s sea pen PCRs from 20150604, using Ultrafree-DA spin columns (Millipore). Transferred gel pieces from storage tubes (1.5mL snap cap tubes) to spin columns. Spun 10,000g, 5mins @ RT. Transferred purified DNA back to original storage tubes. See the sequence_log (Google Sheet) for a full list of the samples and the sequencing plates layouts. Purified DNA was stored @ 4C O/N. Will prepare and submit plates for Sanger sequencing tomorrow.
Currently don’t have sufficient reagents to perform reverse transcription on the entire set of DNased RNA (control and 1hr.heat-shocked O.lurida ctenidia samples). To enable Jake to start testing out some of his primers while we wait for reagents to come in, Steven suggested I generate some cDNA for him to use.
The following DNased RNA samples showed inconsistencies between qPCR reps (one rep showed amplification, the other rep did not) on 20150514:
Isolated RNA from Jake’s Olympia oyster ctenidia, controls, collected on 20150422. Samples had been homogenized and stored @ -80C.
Isolated RNA from Jake’s Olympia oyster ctenidia, 1hr heat shock, collected on 20150422. Samples had been homogenized and stored @ -80C.
Since the previous isolation attempt was unsuccessful (see 20140922), we’re trying a slightly different approach than yesterday.
Isolated gDNA from two C.gigas larvae samples (stored in RNA Later) from Katie Latterhos:
Realized that the PCR performed on 20140828 used the incorrect forward primer! As such, am repeating as before, but with the correct forward primer:
Per Mac’s request, ran a PCR on a set of bisulfite-treated DNA (in her gDNA 2014 box in small -20C):
Isolated RNA from the following samples provided by Jessica Blanchette (stored in RNA later):
Isolated DNA from the following samples, provided by Mackenzie:
Due to the recent poor quality gDNA that has been isolated from C.gigas gonad, I decided to do a quick test using TE for DNA pellet resuspension in hopes that old Buffer EB (Qiagen) or old nuclease-free H2O (Promega) are to blame for the apparent, rapid degradation that I’ve experienced.
Mac’s been having some difficulty getting good quality gDNA from some of her gonad samples, so she asked me to give it a shot.
We’ve had some Illumina sequencing issues with Yanouk’s samples, so I ran the samples out on a 0.8% agarose gel to evaluate the levels of degradation. Loaded 2uL of each sample. Did not load equal quantities of gDNA, due to the lack of available gDNA in the samples we submitted for Illumina sequencing. Added 2uL of H2O to samples 37 & 38 in hopes of having sufficient DNA for visualization on the gel.
Isolated additional geoduck gDNA from the two fresh (now frozen) geoduck’s that Brent provided me with on 20140212 so that we can potentially isolate RNA from the same geoducks to tie in with the DNA Illumina sequencing that Axa will be conducting. Isolated DNA using the DNeasy Blood & Tissue Kit (Qiagen) according to the manufacturer’s protocol (incubated minced siphon tissue at 56C for 3hrs). Eluted with 75uL of ddH2O and spec’d on NanoDrop1000.
After speaking with Axa regarding the DNA concentrations, he would like the DNA from the ethanol-fixed tissue to be more concentrated, and he wants them in ddH2O instead of Buffer AE (from the Qiagen DNeasy Kit). So, I preformed a standard ethanol precipitation. Added 0.1 volumes of 3M sodium acetate (pH = 5.2) [15uL], 2.5 volumes of 100% ethanol [412.5uL] and incubated @ -20C over the weekend.
Since yesterday’s DNA isolation failed to yield sufficient quantity of DNA from the ethanol-fixed samples, I isolated additional DNA from the same samples.
Isolated gDNA from 6 geoduck siphon samples provided by Brent using the Qiagen DNeasy Spin Kit. Samples were as follows:
Samples from yesterday were centrifuged 30mins, 4000g, 4C (fixed angle rotor).
Finally located the remaining half of Emma’s samples. These had already been freeze dried AND pulverized! So, I just had to weigh out ~half of each sample for the glycogen assay.
Samples from yesterday were centrifuged 30mins, 4000g, 4C (fixed angle rotor).
Samples were previously freeze dried overnight and stored @ -20C. To maximize sample homogeneity and, thus, increase accuracy of both assays, all samples were mechanically pulverized in their existing tubes. Approximately half of each sample was weighed and used for the glycogen assay. The remainder of each sample was stored @ -20C.
Ran PCR on lake trout DNA and lake trout bisulfite-converted DNA. Used primers SRIDs: 1551 and 1552. DNA was isolated by Caroline Storer on 4/4/2011 and bisulfite converted on 4/7/2011. Master mix and cycling params are here:
Performed PCR using the primers CG_HK_CDS_2132-2158 (SRID: 1521) and Cg_Hk_CDS_3’_no_stop (SRID: 1519) on pooled C.gigas cDNA (from DATE).
Performed PCR using newly designed primers to amplify the C. gigas hexokinase “promoter” (-2059bp from start) along with a portion of the first exon.
Performed a repeat of the failed PCR from 20130227, but used a pool of cDNA (made from 20110311 C.gigas cDNA) instead of a single sample and changed the annealing temp to 50C.
Performed PCR to amplify the C.gigas hexokinase (ACCESSION#) promoter region (-2059bp) and the CDS without the stop codon. Elimination of the stop codon allows for subsequent cloning into the pBAD-TOPO expression vector, which will incorporate the V5 epitope tag sequence. This tag will be used to distinguish between endogenous hexokinase expression and expression generated from our hexokinase construct.
Performed an RT reaction on pooled herring gonad and liver mRNA from 20091026 for James Raymond at the UNLV. A single RT reaction was performed using 12.75uL (208ng) of the pooled gonad mRNA and 5uL (132.5ng) of the pooled liver RNA, according to our default MMLV (Promega) protocol. After reaction was completed, sample was stored @ -20C and then shipped to James Raymond on 20130214.
Ran qPCR on Halley’s cDNA to see if I could get them to work. She has been getting high levels of fluorescence at the initiation of the qPCR cycling that shouldn’t be there. Master mix calcs and plate layout can be seen here. http://eagle.fish.washington.edu/Arabidopsis/Notebook%20Workup%20Files/20121128%20qPCR%20Layout.jpg
Reverse transcribed the class FISH441 RNA to cDNA. Followed protocol provided on the FISH441 Wiki page, NOT the usual Roberts Lab protocol, with some modifications.
Performed qPCR on Dave’s manila clam larvae DNased RNA from August 2012 using EF1a primers (SR IDs: 1463, 1474).
Photos of oysters are here:
Ran qPCR to test uniformity of Opticon 2, after it was serviced on 20120926.
Performed plasmid isolation on 17 bacterial cultures Emma inoculated yesterday using the QIAprep Spin Mini Kit (Qiagen) using ~1.4mL of culture according to the manufacturer’s protocol. Plasmid DNA was eluted with 50uL of Buffer EB. Tubes were stored @ 4C in the refrigerator in FTR 213.
Took heat-fragmented RNA provided by Emma (see Emma’s Notebook, 7/3/2011) and proceeded to make first strand cDNA, as described in the Eli Meyer protocol for Illumina HiSeq. Master mix calcs are here. Samples were stored @ -20C after the reverse transcription and library construction will be continued tomorrow.
Reconstituted all of the oligos and barcodes for library construction in TE (pH = 8.0) to a final concentration of 100uM. Created 10uM working stocks of all oligos and barcodes. All samples (stocks and working stocks) are stored @ -80C in their own box (Illumina Library Oligos & Barcodes) due to the fact that one of the oligos is an RNA oligo and requires storage at -80C.
After submission of QPX samples to HTGU for Illumina library prep yesterday, I was notified that there was insufficient RNA for the QPX RNA samples. I checked the source RNA on the Roberts Lab NanoDrop1000 and determined that they had high phenol contamination (large peak at 270nm), which results in a large exaggeration in the OD260 absorbance (NanoDrop1000 report[JPEG]; notice terrible OD260/280 ratios; did not save screen shot of absorbance peaks.). As such, the concentrations that Lexie had listed in her notebook for these samples are highly inaccurate and highly inflated. To remove the phenol, I brought all of her QPX RNA samples from 20110504 up to ~200uL with 0.1%DEPC-H2O, added 200uL of chloroform, vortexed for 30s, spun at 12,500g RT for 15mins, and transferred aqueous phase to new tube. Then performed an ethanol precipitation on the aqueous phase. Added 0.1 vols of 3.0M sodium acetate (pH = 5.2), 2.5 vols of 100% EtOH, mixed and incubated at -20C for 1hr. Pelleted RNA by spinning at 16,000g 4C for 15mins.
Submitted samples to HTGU for Illumina sequencing.
Ran qPCR with VtpA primers on cDNA and DNA (from yesterday) of C.gigas larvae to see levels of V.tubiashii compared to their water filter samples (see 20120326). Master mix calcs are here. Plate layout, cycling params, etc can be seen in the qPCR Report (see Results). Used 1uL of cDNA and 100ng (1uL) of DNA as template.
Performed reverse transcription using random primers (Promega) diluted 1:100 (5ng/uL) with 175ng of DNased total RNA. Random primers were used because we will be targeting V.tubiashii RNA instead of eukaryotic RNA. Reverse transcription was performed with M-MLV Reverse Transcriptase (Promega) according to the manufacturer’s protocol. Calcs are here.
Ran qPCR using V.tubiashii VtpA primers (from Elene; no SR ID). Used 0.5uL of each DNased RNA sample, which equals ~40ng of RNA, which would be the equivalent amount of RNA that would end up in a qPCR rxn after cDNA has been made (using 1uL of cDNA). Used the filter DNA extraction from samples #279 from DATE as a positive control. Master mix calcs are here. Plate layout, cycling params, etc. can be found in the qPCR Report (see Results).
Treated 5ug of total RNA (in a 50uL rxn) using Turbo DNA-free (Ambion) according to the “Standard” protocol. Samples were spec’d on the Roberts Lab NanoDrop 1000.
Samples that had been split from earlier today (see the RNA Isolation below) were resuspended in 1mL of DNAzol (MRC). 100ug of Proteinase K (Fermentas) was added to each sample. Samples were incubated at RT, O/N on a rotator. On 20120427 samples were pelleted 10mins, 10,000g, and supe transferred to fresh tube. DNA was precipitated with 0.5mL of 100% EtOH, mixed gently and pelleted 5mins, 5000g. Supe was discarded, pellets were washed with 1mL 75% EtOH, re-pelleted at same speed as previous step, supe discarded and pellets were resuspended in NanoPure H2O. Samples were spec’d on the Roberts Lab NanoDrop1000.
Samples had been stored in RNA Later (Ambion). Samples were pelleted and the RNA Later supe removed. Samples were washed (2x) with 1mL TE (pH = 8.0) to remove excess salt resulting from the RNA Later. Samples were split, roughly equally, into two separate tubes. Samples were pelleted and the supe removed. One tube from each sample was set aside for gDNA isolation using DNAzol (MRC). The other tube was vortexed vigorously in TriReagent (MRC) and the then treated according to protocol. Samples were resuspended in 100uL of 0.1% DEPC-H2O and spec’d on the Roberts Lab NanoDrop 1000.
Ran qPCR on the Taylor water filter DNA extracts from 20120322 using V.tubiashii VtpA primers (provide by Elene; no SR ID?) instead of 16s primers, which failed to produce acceptable results in the melt curves (see 20120323). Additionally, Elene has a standard curve for V. tubiashii (from 1/12/2011) based off of CFUs/mL, which will allow us to quantify theoretical number of V.tubiashii CFUs present in each sample.
Repeated exactly what was done earlier today due to apparent contamination in negative controls.
Ran qPCR on the Taylor water filter DNA extracts from yesterday using V.tubiashii 16s primers (SR IDs: 455, 456). Used RE22 DNA as a positive control, provided by Elene. Master mix calcs are here. All samples were run in duplicate. Plate layout, cycling params, etc can be found in the qPCR Report (see Results).
Extracted DNA from the following water filter samples using the Qiagen DNeasy Blood & Tissue Kit: