Feb 09, 2023

Public workspacePreparing multiplexed WGS/MetaG libraries with the Illumina DNA Prep kit for the Illumina NextSeq or MiSeq

DOI
dx.doi.org/10.17504/protocols.io.5qpvob31bl4o/v1
  • 1Integrated Microbiome Resource (IMR), Dalhousie University
André M Comeau
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DOI: dx.doi.org/10.17504/protocols.io.5qpvob31bl4o/v1
Protocol Citation: André M Comeau, Gina V Filloramo 2023. Preparing multiplexed WGS/MetaG libraries with the Illumina DNA Prep kit for the Illumina NextSeq or MiSeq. protocols.io https://dx.doi.org/10.17504/protocols.io.5qpvob31bl4o/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: March 07, 2022
Last Modified: February 09, 2023
Protocol Integer ID: 59164
Abstract
The preparation of (meta)genomic libraries using the Illumina Nextera Flex (now "DNA Prep") kit with CD/UDI indices at the IMR.

Based upon Illumina protocol 10000000254 16 v10 (Aug.2021).
Materials
The following materials list contains those consumables+quantities used specifically at the IMR to complete the present protocol for 192 metagenomes (2 plates) on the NextSeq2000.

Pre-Library Sample Quantification
Quant-iT 1X HS dsDNA Kit (1000 samples)
Optical plate
PCR microplates 96-well Bio-Rad
PCR microplates sealing film Bio-Rad
Tips ClipTip 20
Tips ClipTip 200
Tips ClipTip 300
Reservoir
UltraPure water

Illumina Library Preparation
Nextera Flex DNA Sample Prep Kit (96 samples)
IDT for Illumina DNA/RNA UD Indexes Set A, Tagmentation (96 Indexes, 96 Samples)
IDT for Illumina DNA/RNA UD Indexes Set B, Tagmentation (96 Indexes, 96 Samples)
PCR microplates 96-well Bio-Rad
PCR microplates sealing film Bio-Rad
Manual p1000 tips
Tips ClipTip 20
Tips ClipTip 200
PCR tube strips (Axygen, 8 x 0.2 mL)
15 mL Falcon tubes
50 mL Falcon tubes
Ethanol
Reservoir
UltraPure water

Post-Library Quantification and Pooling
Quant-iT 1X HS dsDNA Kit (1000 samples)
Optical plate
PCR microplates sealing film Bio-Rad
Tips ClipTip 20
Tips ClipTip 300
PCR tube strips (Axygen, 8 x 0.2 mL)
1.5 mL Eppendorf tubes
Reservoir

Loading and Sequencing
PhiX Control Kit v3 (at least for 5 stocks)
NextSeq 2000 P3 Kit 300 cycle
Quant-iT 1X HS dsDNA Kit (1000 samples)
Qubit Tubes
1.5 mL Eppendorf tubes
Various manual pipette tips
Prepare and Quantify gDNA
Prepare and Quantify gDNA
Using two 96-well PCR plates, prepare Amount30 µL of 1:10 dilution using nuclease-free water (NFW) for up to 192 gDNA samples.

Quantify Amount5 µL of the 1:10 diluted gDNA using the Invitrogen Quant-iT dsDNA HS assay kit on the fluorescent 96-well plate reader.

Note
Sample concentrations are required for calculating equal molar DNA ratios for library preparation input. Our standard practice is to add the appropriate volume of each sample so that Amount5 ng of gDNA are added to each sample well.


Perform Illumina DNA Prep Kit Library Preparation
Perform Illumina DNA Prep Kit Library Preparation
7m
7m
Follow the library preparation instructions as described for the Illumina DNA Prep kit (https://support.illumina.com/content/dam/illumina-support/documents/documentation/chemistry_documentation/illumina_prep/illumina-dna-prep-reference-guide-1000000025416-10.pdf) with the below modifications.

Note
We were originally doing up to 96 samples (1 plate) on the NextSeq550 and so using the Nextera DNA CD Indexes (96 Indexes) for indexing. However, we have now increased to up to 192 samples (2 plates) on the NextSeq2000 and so are now using the IDT for Illumina DNA/RNA UD Indexes Set A and B, Tagmentation (96+96 Indexes) for indexing.

Amplify Tagmented DNA: use 12 PCR cycles (based on 5 ng of input DNA).
Final library clean-up: wash beads with 80% ethanol as described, remove excess ethanol and allow beads to dry on the magnet for Duration00:05:00 . Remove the plates from the magnet and add Amount60 µL of Resuspension Buffer (RSB) to the beads. Pipette mix until thoroughly resuspended and incubate at room temperature for Duration00:02:00 . Place the plates back on the magnet until clear and transfer Amount55 µL of the supernatant into new 96-well plates.


7m
Quantify and Pool Final Library
Quantify and Pool Final Library
Quantify Amount5 µL of each tagmented, amplified and purified sample using the Invitrogen Quant-iT dsDNA HS assay kit on the fluorescent 96-well plate reader.
Pool equal molar amounts of up to 192 samples in a 1.5 mL microcentrifuge tube.
Dilute pooled library with RSB to the recommended loading concentration specific to the intended sequencer per Illumina's guidelines.