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Sample and Library Prep

This document provides a protocol for sample/library preparation for nCoV-2019 sequencing including cDNA preparation using reverse transcription, primer pool multiplex PCR, PCR product merging and dilution, native barcoding with barcode addition, cleanup steps using SPRI beads, adapter ligation, and final quantification.

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Shavana Rajkumar
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21 views4 pages

Sample and Library Prep

This document provides a protocol for sample/library preparation for nCoV-2019 sequencing including cDNA preparation using reverse transcription, primer pool multiplex PCR, PCR product merging and dilution, native barcoding with barcode addition, cleanup steps using SPRI beads, adapter ligation, and final quantification.

Uploaded by

Shavana Rajkumar
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
Available Formats
Download as PDF, TXT or read online on Scribd
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Sample/ library preparation for nCoV-2019 sequencing

UWI COVID-19 IMPACT adaptation of nCoV-2019 sequencing protocol v3


(LoCost)V.3; Josh Quick; University of Birmingham

cDNA preparation (RT)_ For each sample enzyme added to tubes in Room 7, Samples added in Room
8

Components Volume
LunaScript RT SuperMix (5X) 2 µL
Template RNA 8 µL
Total 10 µL
Mix and pulse spin down

Incubate reaction as follows:

25 °C for 2:00 mins

55 °C for 10:00 mins

95 °C for 1:00 mins

Hold at 4 °C

Primer pool multiplex PCR_Enzyme/ Primers prep in Room 7, Sample added in Room 8
Set up the two PCR reactions per sample as follows in tubes.

Components Reaction 1 and 2 For 26 reactions


Q5 Hot Start High-Fidelity 2X Master Mix 12.5 µL 325 µL
Pool 1 (10µM)/ Pool 2 (10µM) primers 4 µL 104 µL
Nuclease-free water 6 µL 156 µL
Total 22.5 µL
Gently mix and pulse spin

Add 2.5 µl cDNA product to each of the PCR reactions. Gently mix and pulse spin down.

PCR reaction as follows:

Heat Activation at 98 °C for 30 seconds

Denaturation at 98 °C for 15 seconds for 30 cycles

Annealing at 65 °C for 5:00 mins for 30 cycles

Hold 4 °C
PCR merge and dilution _ Room 8
For each sample create one dilution by, merging the same samples (different pool products).

Components Volume
Pool 1 PCR reaction product 4 μL
Pool 2 PCR reaction product 4 μL
Nuclease-free water 42 μL
Total 50 ul

Native barcoding_ For each sample set up the enzyme and barcode tubes in Room 7, add PCR product
(diluted) Room 8

STEP 1 – End prep

Component Volume For 26 reactions


Ultra II End Prep Reaction Buffer 1.2 μL 31.2 μL
Ultra II End Prep Enzyme Mix 0.5 μL 13 μL
Nuclease-free water 5 μL 130 μL
PCR dilution from previous step 3.3 μL
Total 10 ul

Incubate at 25 °C for 15 minutes

Incubate at 65 °C 15 minutes

Incubate at 4 °C for 1 minute and hold at same temperature

STEP 2 – Adding barcodes

Components Volume For 26 reactions


End-preparation reaction mixture 1 μL
Blunt/TA Ligase Master Mix 2 μL 52 μL
Nuclease-free water 6 μL 156 μL
NBXX barcode 1.25 μL
Total 10.25 μL

Incubate at 25 °C for 20 minutes

Incubate at 65 °C for 10 minutes

Incubate at 4°C for 1 minute and hold at same temperature

Pooling the pooled


In a new 1.5 mL microcentrifuge tube, pool all one-pot barcoding reactions together. When processing
1-24 samples pool all 10 μl from each native barcoding reaction. (240 ul total)

Clean-up of the sample

FIRST CLEAN-UP
1) Add 96ul (0.4x volume) of SPRI beads to the one-pot barcoding reaction of 240ul and mix gently
by either flicking or pipetting. Mix by vortexing and pulse centrifuge to collect all liquid at the
bottom of the tube. Incubate for 5 minutes at room temperature.

2) Place on magnetic rack and incubate for 2 minutes or until the beads have pelleted and the
supernatant is completely clear. Carefully remove and discard the supernatant, being careful not
to touch the bead pellet.

3) Add 250 μl SFB and resuspend beads completely by pipette mixing. Pulse centrifuge to collect all
liquid at the bottom of the tube and place on the magnet. Remove supernatant and discard.
Pulse centrifuge and remove any residual SFB. REPEAT OPTION

4) Add 200 μl of room-temperature 70 % ethanol to bathe the pellet. Carefully remove and discard
ethanol, being careful not to touch the bead pellet. Pulse centrifuge to collect all liquid at the
bottom of the tube and carefully remove as much residual ethanol as possible using a P10
pipette. With the tube lid open incubate for 1 minute or until the pellet loses its shine (if the
pellet dries completely it will crack and become difficult to resuspend).

5) Resuspend pellet in 30 μl 10 Milimolar (mM) Tris pH 8.0, mix gently by either flicking or pipetting
and incubate for 2 minutes.

6) Place on magnet and transfer sample to a clean 1.5 mL microcentrifuge tube ensuring no beads
are transferred into this tube.

QUANTIFY
7) Quantify 1 μl of the barcoded amplicons by adding 1ul and 199ul dye solution (Qubit dsDNA HS)
to a Qubit assay tube. Mix sample vigorously by vortexing for 5 seconds then pulse centrifuge to
collect the liquid. Allow tubes to incubate at room temperature for 2 minutes before
proceeding. Read on the Qubit Fluorometer using the dsDNA selection. The desired total
concentration is about 30 ng.

Adapter ligation and clean-up with SFB

1) In a new 1.5 μl microcentrifuge tube, set up the following AMII adapter ligation reaction and
incubate at room temperature for 20 minutes.
Components Volume
NEBNext Quick Ligation Reaction Buffer (5X) 10 μL
Adapter Mix (AMII) 5 μL
Quick T4 DNA Ligase 5 μL
Barcoded amplicon pool 30 μL
Total 50 μL

2) Add 50 μl (1:1) of SPRI beads to the sample tube and mix gently by either flicking or pipetting.
Pulse centrifuge to collect all liquid at the bottom of the tube. Incubate for 5 minutes at room
temperature.

3) Place on magnetic rack and incubate for 2 minutes or until the beads have pelleted and the
supernatant is completely clear. Carefully remove and discard the supernatant, being careful not
to touch the bead pellet.

4) Add 50 μl SFB and resuspend beads completely by pipette mixing. Pulse centrifuge to collect all
liquid at the bottom of the tube. Remove supernatant and discard. Pulse centrifuge and remove
any residual SFB. REPEAT OPTION

5) Add 15 μl EB (ONT) and resuspend beads by pipette mixing. Incubate at room temperature for 2
minutes.

6) Place on magnetic rack until clear. Transfer final library to a new 1.5mL Eppendorf tube.

7) Repeat the quantification as previously described. The desired total concentration is about 15ng
for the final library.

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