Projects

Miscellaneous

FastQC-MultiQC - Additional C.gigas WGBS Sequencing Data from Genewiz Received 20190501

  • ~1 min read

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.

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Data Analysis - C.virginica MBD Sequencing Coverage

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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.

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Metagenomics Gene Prediction - P.generosa Water Samples Using MetaGeneMark on Mox to Compare pH Treatments

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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.

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Metagenome Assemblies - P.generosa Water Samples Trimmed HiSeqX Data Using Megahit on Mox to Compare pH Treatments

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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:

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RNA Isolation and Quantification - Crab Hemolypmh Using Quick-DNA-RNA Microprep Plus Kit

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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:

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Transcriptome Assembly - Geoduck Tissue-specific Assembly Larvae Day5 EPI99 with HiSeq and NovaSeq Data on Mox

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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.

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Data Management - Data Migration and Drive Expansion on Gannet

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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.

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Genome Assessment - BUSCO Metazoa on P.generosa v071 on Mox

  • 6 min read

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).

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Data Wrangling - CpG OE Calculations on C.virginica Genes

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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.

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Methylation Analysis - C.virginica

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This is a quick and dirty (i.e. no adaptor/quality trimming) assessment of all of our Crassostrea irginica bisulfite sequencing efforts to date in order to get a rough idea of the methylation mapping, per this GitHub issue. Ran Bismark on Mox on a series of subset of the reads:

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DNA Isolation and Quantification - Ronit’s C.gigas Ploidy Ctenidia

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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.

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qPCR - Relative mitochondrial abundance in C.gigas diploids and triploids subjected to acute heat stress via COX1

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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.

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FastQC and Trimming - Metagenomics (Geoduck) HiSeqX Reads from 20180809

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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.

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Installation - Microsoft Machine Learning Server (Microsoft R Open) on Emu/Roadrunner R Studio Server

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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.

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Mox - Password-less SSH!

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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.

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Total Alkalinity Calculations - Yaamini’s Ocean Chemistry Samples

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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.

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FAIL - Missing Data on Owl!

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Uh oh. There appears to be some data that’s been removed from Owl. I noticed this earlier when trying to look at some of Sean’s data. His data should be in a folder with his name in Owl/scaphapoda

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RNA Isolation - Olympia oyster gonad tissue in paraffin histology blocks

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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.

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RNA Isolation - Olympia oyster gonad tissue in paraffin histology blocks

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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

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Data Received – Jay’s Coral epiRADseq - Not Demultiplexed

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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.

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Oyster Sampling - Olympia Oyster OA Populations at Manchester

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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:

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Computing - Amazon EC2 Cost “Analysis”

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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).

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Dissection - Frozen Geoduck & Pacific Oyster

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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.

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Opticon2 Calibration

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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.

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Reverse Transcription - O.lurida DNased RNA Controls and 1hr Heat Shock

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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.

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Gel Purification - Olympia Oyster and Sea Pen PCRs

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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.

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DNA Isolation - Test Sample

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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.

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DNA Quality Check - Yanouk’s Oyster gDNA

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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.

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DNA Isolation - Geoduck

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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.

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DNA Precipitation - Geoduck DNA from 20140213

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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.

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DNA Isolation - Geoduck

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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.

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DNA Isolation - Geoduck

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Isolated gDNA from 6 geoduck siphon samples provided by Brent using the Qiagen DNeasy Spin Kit. Samples were as follows:

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Gylcogen and Carboyhydrate Assays - Emma’s C.gigas Whole Body Samples

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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.

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PCR - Lake Trout C1q

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Repeated PCR from 20130919, but with a newly designed reverse primer. Primers were designed using MethPrimer (SR IDs: 1553, 1555). Primers were designed using MethPrimer:

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PCR - Lake Trout C1q

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Repeated PCR from 20130919, but with a newly designed set of primers that targets the desired region of C1q for sequencing (SR IDs: 1553, 1554). Primers were designed in Geneious.

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PCR - Lake Trout C1q

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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:

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PCR - Hexokinase Partial CDS

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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).

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PCR - Hexokinase Promoter and CDS

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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.

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Reverse Transcription - Herring RNA from 20091026

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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.

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Reverse Transcription - FISH441 RNA

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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.

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Minipreps - Emma’s Illumina Library Cloning

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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.

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Oligo Reconstitution - Illumina RNAseq Library Oligos and Barcodes

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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.

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Chloroform Clean Up - Lexie’s QPX RNA from 20110504

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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.

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Reverse Transcription - DNased C.gigas Larval RNA from 20120427

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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.

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qPCR - Check DNased RNA from Earlier Today for Residual gDNA

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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).

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gDNA Isolation - C.gigas Larvae from Taylor Summer 2011

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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.

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RNA Isolation - C.gigas Larvae from Taylor Summer 2011

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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.

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qPCR - Taylor Water Filter DNA Extracts from 20120322

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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.

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