Grace’s Notebook: Gave Pooled Sample to the NWGC for Library Prep and RNA-Seq!

Today I finished speed vac-ing (medium heat) the pooled sample. It ended up being too low of volume (14.1ul), so I added 40.9ul of 0.1% DEPC-treated H20. I ran 2ul of the sample on Qubit (RNA HS), and got a reading of 20.4 ul (1,081 ng RNA in the sample)!!!! We FINALLY have a sample to send off for library prep and sequencing! After getting info from NWGC (Chris in the Nickerson Lab at Foege) I put the sample on dry ice and walked it over! It is now in their hands until we get the data back. 🙂

Sample prep

Yesterday wasn’t enough time on the speed vac. So I put it back on (on medium) at 10:10 am. I took it off at 1pm.

I checked the volume of the sample by sucking it all up in a pipet tip (set to 50ul). Then, I decreased the volume on the pipet tip until the liquid was at the tip. It showed that the volume was 14.1 ul. It needs to be at least 50ul. So I sucked the sample back up with the pipet set at 14.1 ul. It all fit.

Then I added 40.9ul of 0.1% DEPC-treated H20 so the final volume was 55ul and vortexed it for 10s.

I then used 2ul of the sample to run on the Qubit (RNA HS) to check the RNA quantity. It read as having 20.4ng/ul of RNA! Meaning that in the sample, we had ~ 1,081 ng of RNA!!! (NWGC requires a minimum of 1000ng of RNA in a sample at least 50ul in volume).

I contacted NWGC to get info on what all they needed before I walked the sample over.

I brought it over on dry ice and handed it to Chris Frazar in the Nickerson Lab in Foege at 2:45pm.

He emailed me and Steven (I cc’ed Sam) to get more info on the sample and what kind of sequencing we want. More information on that to come. They will do the library prep and the sequencing.

I am beyond excited we finally have a sample at NWGC!!

Next steps

  • Try Tri-reagent method of RNA isolation once the lyophilizer is fixed
  • Perform qPCR assay to test shellfish primers (GitHub Issue #353)

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Roberto’s Notebook: Problem with Stringtie IDs.

Using the program Stringtie for the transcript abundance estimation for libraries from 2 thermal-resistant (TR) and 2 thermal-susceptible (TS) oyster families exposed to oscillatory thermal challenge during 30 days. The program assigned different gene IDs (different from C. gigas gene IDs) specifically in merge step in the output file (/Volumes/toaser/roberto/Hisat_results/stringtie_results/stringtie_merged.gtf) but it has the reference ID (CGI_10000005 for example). As Steven suggested, using finder on stringtie_merged.gtf file, I found the missing CGI gene IDs in the gene expression table (where only 4379 genes had CGI IDs from 60643 total expressed genes).
Doing a test, the stringtie ID “MSTRG.21417” corresponds to DNMT1 gene CGI_10021920 (https://www.uniprot.org/uniprot/K1QQH9). It is differentially expressed between TR (samples Os13, Os14, Os15, Os16, Os17 and Os18) and TS families (Os1, Os2, Os3, Os10, Os11 and Os12) at day 30.
DNMT1 dene expression day 30

Where phenotype looks with isoform preferences:
DNMT1 phenotype expression day 30

As is the case for the isoform #2 (considering up to down in the figure) located in:
scaffold1862: transcript_position: 618219-640790 / gene_id “MSTRG.21417”; transcript_id “MSTRG.21417.4”; ref_gene_id “CGI_10021920” with 34 exons is more present in TR. This could suggest… 🙂

Sam’s Notebook: Assembly Stats – Geoduck Hi-C Final Assembly Comparison

0000-0002-2747-368X

We received the final geoduck genome assembly data from Phase Genomics, in which they updated the assembly by performing some manual curation:

There are additional assembly files that provide some additional assembly data. See the following directory:

Actual sequencing data and two previous assemblies were previously received on 20180421.

All assembly data (both old and new) from Phase Genomics was downloaded in full from the Google Drive link provided by them and stored here on Owl:

Ran Quast to compare all three assemblies provided (command run on Swoose):

  /home/sam/software/quast-4.5/quast.py \ -t 24 \ --labels 20180403_pga,20180421_pga,20180810_geo_manual \ /mnt/owl/Athaliana/20180421_geoduck_hi-c/Results/geoduck_roberts results 2018-04-03 11:05:41.596285/PGA_assembly.fasta \ /mnt/owl/Athaliana/20180421_geoduck_hi-c/Results/geoduck_roberts results 2018-04-21 18:09:04.514704/PGA_assembly.fasta \ /mnt/owl/Athaliana/20180822_phase_genomics_geoduck_Results/geoduck_manual/geoduck_manual_scaffolds.fasta 

Grace’s Notebook: Speed Vac New Pool of 15 Samples

Today I speed vac-ed the new pool of 15 samples for a little over three hours. There is still more than 50ul in the tube, so I’ll put it back in the speed vac as soon as I am in tomorrow morning.

The pooled sample is 150ul (15 samples, each 10ul).
Sam and I went over to the Speed Vac in FSH. At 10:50am, it was started on medium heat.

At 2:10pm, there was still too much liquid. As soon as I am in tomorrow am, I’ll put it back in the speed vac.

Put the sample in (cap open) a slot that has a little bit of paper towel stuffed in the bottom. Close lid. Turn on the machine. Turn the heat to medium. After it runs for a bit, turn on the vacuum (turn yellow valve marked “Vac” toward the machine). Then open up the vacuum by turning the blue dial on top of the machine to “open” (to the right).

To open it later, close the vacuum (blue dial) and then wait til it stops spinning.

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