Peromyscus Project Useful Links and Knowledge - erynmcfarlane/StatsGenLabProtocols GitHub Wiki

Google Sheets

Initial doc for planning what to buy for the lab: https://docs.google.com/spreadsheets/d/15Zq9YF15BgaULsAEMTxt-KhYwsmoW4YTU3X_HqN_CNI/edit?usp=sharing

Current inventory and items on order doc: https://docs.google.com/spreadsheets/d/14k8RZ6WfAm7ok0fYLOaNPk9Iovx0UH8wcM-c8Npx_g4/edit?usp=sharing

Amanda M's W24 DNA Extraction and Lib Prep Schedule: https://docs.google.com/spreadsheets/d/1i1opjABjKlnYr8vLKli01mFg2Rp386hwG71BKtpY2mk/edit?usp=sharing

Google Drive

QUBS Data, including Qubit data on completed extractions, barcode layout file, and plating information: https://drive.google.com/drive/folders/1UG6Tm5cR1D7DZi73ogAXsmaZZZDOschE?usp=sharing

Amanda M's thoughts on organization for the Peromyscus project

Metadata Management

All metadata for the Permyscus samples are kept in the PEROMYSCUS_DNA_AM Excel sheet that was initially sent to us by Vincent Careau from UOttawa (who gave us the mouse samples from QUBS).

After extracting DNA from each sample and measuring it on the Qubit, I pulled the Qubit data from the machine onto a USB key, added in each sample's DNA numbers, then uploaded the file to the Google Drive, in the Qubit_Data folder. I then copied the concentration data into the PEROMYSCUS_DNA_AM Excel sheet, and also added the date of extraction, the name of the person who did the extraction, and any notes.

For library prep data, I added to the metadata sheet which library the sample is in, which restriction/ligation and PCR plate the sample is on and what well it's in, and which barcode plate was used. Each R/L plate should be drawn in the lab book prior to plating, so that there's a paper copy that can be looked at while in the lab, in addition to the excel file.

After sequencing data is received and it's time to demultiplex the samples, a barcode key will need to be created that contains the sample number (which will become the name of the fasta file for the ind), and the barcode. This can be generated using the barcode plate and well position from the metadata file and the barcodes and well position from annealed_barcode_layout_march2024.csv file, in the Google Drive.

R/L and Barcode plates

When I choose which barcode plate to use for which R/L plate, I typically try to go in order i.e. R/L plate 1 = Barcode plate 1. This wasn't possible initially as we started R/L for library 1 before receiving all the barcode stock plates from Liz Mandeville. But as a rule, I do odd numbered R/L plates with odd numbered barcode plates, so that it's easier to intuitively know what goes with what and prevent mix ups. Following this rule for the remainder of library 1, the two partial plates 5 & 7 should go with barcode plate 3, and the full R/L plate 6 should go with barcode plate 4.

It's best if the barcode plates are remade annually, to prevent unequal evaporation in the freezer from concentrating some of the wells more than others. Otherwise, don't touch the stock plates in the freezer. Don't even look at them. Liz and Eryn split on barcodes, so plates 1AB, 2AB, and 3AB are in short plates, wrapped in bubble wrap, in ziploc bags. Plates 4AB, 5AB, and 6AB are ones we've ordered from IDT, so they're in deep plates, with lids held on by elastics. All plates have foil covers on them, which should be replaced with fresh ones each time they're opened. As of Apr. 30th 2024, we're out of foils and we've previously just borrowed them from the Mandeville lab, so a supplier will need to be found for them. You must always use filter tips when working with the barcodes, both stock plates and working plates!!! Specifically, we need the XL (long) 10ul filter tips for working with the deep-well plates, and we don't keep many of them in the lab as we don't use them for much else, so a case of 10 boxes needs to be ordered before remaking the working barcode plates.

PCR

Planning how to pool individuals for PCR can be a bit of mental gymnastics. This is covered in the lib prep protocol, but I wanted to note on it here, too, for more detail. Centrifuge plates, then pipette restriction-ligation product to a total of 3uL per well into a new 96-well PCR plate. Multiple plates can be pooled into one, though it is difficult to accurately pipette less than 1uL. Additionally, it is thought that duplicating each PCR plate will adjust for any unequal amplification. Example: if 6 plates are being pooled, pipette 1uL from each well to a new plate (6ul total per well), then pipette 3uL from each well of the new plate to a second plate.

This would be simple if all 6 plates were mice, then we would have only 2 PCR plates. Because we want to do PCR (and size selection with the Pippin) separately for the mice and grass (and other species in the future like butterflies), we will have to pool individuals differently. I would propose:

Grass - Collapse in half, to go from 14 columns to 7, then duplicate. So take 3ul of columns 1 & 2, put in column one on a new plate. Again with columns 3 & 4 into column 2 on a new plate, and so forth. This yields 6ul in each well, for 7 columns, on a new plate. Then to duplicate, divide this in half by taking 3ul of each well and placing in a new plate. You should end with 2 identical plates with 3ul of liquid per well.

Mice - We'll collapse down to one plate, then duplicate. Take 1.2ul from each well and add to a new plate, for a total of 6ul per well. The final two columns of the new plate will have only 4.8ul total, as the last two columns of plate 5 are empty. Then to duplicate, take 3ul from each well and plate into a new plate. Remember to adjust the pipette down to 2.4ul for the final two columns!!!!! These final two columns will have a relative excess of reagents compare to the other 10 columns which have a full 3ul, but I think this is better than over representing some individuals by adding more R/L product from these inds.

This way, there will be 4 PCR plates total for all 6 of the initial R/L plates, and PCR can be done across 2 days. Both PCR plates can be combined into two tubes (one for each species), and could be run on one Pippin cassette together, 2 lanes for each species, IF tape station shows that the same size region should be selected for both species. If it looks better to choose differently sized fragments, then they'll need to be ran on 2 separate cassettes.