Sent 10μg of the geoduck gDNA I isolated earlier today to Illumina on dry ice via FedEx Standard Overnight service.
Sent the following samples to Illumina for possible selection in a new pilot sequencing platform they’re working on.
The 12 samples will be used for RNAseq for genome annotation – numbers indicate desired sequencing priority.
Juvenile and larval samples were from Hollie Putnam (see links below for more info).
Other tissue was from a single, adult geoduck, collected by Brent & Steven on 20150811.
- Juvenile OA exposure (super low) (EPI_115, EPI_116)
- Juvenile ambient exposure (ambient treatment) (EPI_123, EPI_124)
- Larvae day 0 (EPI_74, EPI_75)
- Larvae day 5 (EPI_99)
- Crystalline style
- Byssus gland
- Labial palps
- Juvenile OA exposure – low treatment (EPI_107, EPI_108)
In addition to the above 12 samples, ~1.5μg of geoduck gDNA (isolated this morning) was sent.
Hollie Putnam asked me to help her get samples ready for submission for Illumina sequencing at Genewiz.
She had previously prepared reduced representation bisulfite libraries on 20161215.
She also prepped a whole genome library on 20161201 – specifically sample EPI_135 WG.
She needed these samples combined in to four separate pools. However, Pool 5 was to be pooled with a total of five samples, including EPI_135 WG. She asked that the EPI_135 WG sample make up 50% of the DNA in the pool.
Using her previously determined sample concentrations, I pooled the libraries in equal quantities.
Calculations (Google Sheet): 20161219_hollie_library_pool_calcs
The pool volumes are high and, the calculated pool concentrations are low. Due to time limitations on our end, it was not feasible for me to SpeedVac these down to achieve the target concentration of 10nM. I’ve notified Hollie and asked her to see if Genewiz will perform that service.
Samples were shipped on dry ice to Genewiz via FedEx Standard Overnight.
|SAMPLE||VOLUME FOR 10ng (μL)|
Samples were sent to Genewiz on dry ice via standard overnight FedEx.
Pooled the libraries into a single sample for sequencing on Illumina HiSeq2500 by Genewiz.
Here’s the list of samples that were pooled:
|SAMPLE NAME||LIBRARY NAMES||INDEX 1 (i7)||LENGTH (bp)||INDEX 2 (i5)||LENGTH|
|SJW_Oly_2bRAD||Oly RAD 02||CGTGAT||6||ATGCAT||6|
|SJW_Oly_2bRAD||Oly RAD 03||ACATCG||6||ATGCAT||6|
|SJW_Oly_2bRAD||Oly RAD 04||GCCTAA||6||ATGCAT||6|
|SJW_Oly_2bRAD||Oly RAD 06||TGGTCA||6||ATGCAT||6|
|SJW_Oly_2bRAD||Oly RAD 07||CACTGT||6||ATGCAT||6|
|SJW_Oly_2bRAD||Oly RAD 08||ATTGGC||6||ATGCAT||6|
|SJW_Oly_2bRAD||Oly RAD 14||GATCTG||6||ATGCAT||6|
|SJW_Oly_2bRAD||Oly RAD 17||TCAAGT||6||ATGCAT||6|
|SJW_Oly_2bRAD||Oly RAD 23||CTGATC||6||ATGCAT||6|
|SJW_Oly_2bRAD||Oly RAD 30||AAGCTA||6||ATGCAT||6|
Combined 40ng of all samples, except Oly RAD 30. Used only 20ng of Oly RAD 30 because it was the only sample that produced a single peak in qPCR melt curve analysis (i.e. no primer dimer). As such, it’s a rough assumption that the qPCR quantitation of all the other samples is twice as high as they should be due to the contribution of primer dimer amplification.
Calculations for pooling can be seen here (Google Sheet): 20151117_RAD_qPCR_data
The full list of samples (and the individual samples/libraries/indexes) submitted to Genewiz for this project by Katherine Silliman & me can be seen here (Google Sheet): White_BS1511196_R2_barcodes
We opted to go with ZymoResearch for this project because they could do the bisulfite treatment and finish the sequencing by the end of January.
Submitted the following 18 Ostrea lurida MBD-enriched gDNA samples to ZymoResearch for bisulfite treatment and subsequent Illumina sequencing (50bp, single read):
The samples will be bisulfite treated, Illumina libraries constructed, multiplexed, and this multiplexed library will be sequenced three times.
Yep, BGI still needs more gDNA for the Olympia oyster genome sequencing project. Samples have been quantified via dye-based fluorescence, as opposed to the NanoDrop, so our quantities should be more accurate and in-line with what BGI will also find.
Submitted three separate isolations, just in case the quality of one was unacceptable, I didn’t want to pool the samples and have that one bad apple ruin the entire batch.
In total, submitted ~31μg.
Samples were shipped on dry ice with the appropriate paperwork required by BGI (sample declaration letter).
Assigned BGI Lot: 1512021005
Sent Olympia oyster tissue samples to BGI for genotype-by-sequencing (GBS). Tissues were shipped FedEx standard overnight on dry ice.
Tissues were collected by Brent, Steven & Jake on 20151124 and were frozen @ -80C.
36 samples from each of the three populations were collected. Only 32 samples from each population (total 96 samples) will be sequenced, but wanted to send extras from each population in case any were of poor quality.
Sample submission sheet is here (Google Sheet): 20151130_BGI_GBS_tissue_submission
Previous shipment of gDNA proved to be of insufficient quantity when assessed by BGI, so needed to isolate more.
Shipped the pooled gDNA we’ve been accumulating to BGI to contine the geoduck genome sequencing project.
Sample was shipped on dry ice with the appropriate paperwork required by BGI (sample declaration letter).
Assigned BGI Lot: 1510071002