Sep 12, 2022
Final QC, Pooling and Sequencing
- 1UC San Diego;
- 2Palmer Lab
Protocol Citation: Katarina A Cohen, Khai-Minh H Nguyen, Oksana Polesskaya, Abraham Palmer 2022. Final QC, Pooling and Sequencing . protocols.io https://dx.doi.org/10.17504/protocols.io.yxmvmnw29g3p/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: May 01, 2022
Last Modified: September 12, 2022
Protocol Integer ID: 61760
Abstract
This protocol is conducted after a set of libraries are completed and ready to quantify and pool. This protocol outlines the final steps before submitting for sequencing.
Materials
Equipment
- PC Running Excel
- Pipette 1-10uL
- Pipette 20-200uL
- Bioanalyzer
- Qubit Flourometer
Reagents
- Ultrapure Distilled, Nuclease Free Water
Consumables
- Pipette Tips
- 1.5mL Eppendorf Tubes
- Qubit Assay
- Tapestation D1000
Protocol materials
Ultrapure Distilled, Nuclease Free Water
In Materials and 2 steps
Before start
Ensure all libraries that will be pooled are uniquely indexed.
Library QC
Library QC
- Quantify purity and concentration of library with Nanodrop and a Qubit Assay
- Obtain average fragment size of library with Tapestation (D1000 Assay)
- Libraries should have an average fragment size between 420bp - 650bp.
- 260/280 should be around 1.80 - 2
- 260/230 should be around 2-2.2.
- We have been able to get good data from libraries with relatively poor nanodrop purities.
- Qubit concentrations can widely range. We get a range from 10ng/ul - 60ng/ul
Pooling
Pooling
Download 
Pooling Template.xlsx
Pooling Template.xlsx - Increase "Target Vol (uL) per Sample" if any Sample Vol is lower than 1ul
- Use Ultrapure Distilled, Nuclease Free Water when adding water to final pool
Enter library name in Column A "Sample Name"
Enter qubit concentrations in column B
Enter average fragment size of library in column C for each library.
Increase "Target Vol (uL) per Sample" if any Sample Vol is lower than 1ul
Label new tube as Riptide Pool ## , and Date. Add calculated volume of water (shown in cell I6). Add calculated volumes of sample shown in column E.
- Use Ultrapure Distilled, Nuclease Free Water when adding water to final pool
Checking Pooling
Checking Pooling
RECOMMENDED OPTIONAL STEP
Check pooling with an illumina MiSeq run
- % Reads Identified for each library shouldn't vary more than 30% from each other.
Sample-Barcode List
Sample-Barcode List
YYYY-MM-DD-Flowcell Sample-Barcode list.xlsx - Download the file above
- Check Twist Bioscience site for updates to the sample barcodes used for the Twist 96-Plex Kit.
Open the library file created in the "EPMotion - Normalization and Randomization".
- Go to the "Sample_Randomization" Tab
When the "Sample_Randomization" tab is opened, copy and paste the randomized Transponder ID's into Column A of the Flowcell Sample-Barcode list.
- NOTE: If less than 96 samples are processed in a library, delete the unfilled rows within that 96 library set. (Essentially, you want to delete the sample barcodes that are not associated with a sample).
Transfer any comments from the library file into Column G of the Sample barcode list
- Ensure that the comment matches with the correct sample ID.
Enter the PCR index barcode used for that particular library (ex. 1,2,3,4,5...) in Column D
Enter Library name in Column E
Submitting for Sequencing
Submitting for Sequencing
Submit 30ul of Pool
- Platform: NovaSeq S4
- Run Type: PE150
You can find the updated inline i7 and i5 index sequences on Twist Bioscience Site