Dec 07, 2022
Version 1
RAPIDprep: A simple, fast protocol for RNA metagenomic sequencing of clinical samples V.1
This protocol is a draft, published without a DOI.
- Karan Kim1,2,
- Racheltulloch1,2,
- John-Sebastian Eden1,2
- 1Westmead Institute for Medical Research;
- 2University of Sydney
Protocol Citation: Karan Kim, Racheltulloch, John-Sebastian Eden 2022. RAPIDprep: A simple, fast protocol for RNA metagenomic sequencing of clinical samples. protocols.io https://protocols.io/view/rapidprep-a-simple-fast-protocol-for-rna-metagenom-cjveun3e
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: November 29, 2022
Last Modified: December 07, 2022
Protocol Integer ID: 73350
Keywords: metagenome, NGS, virome, rapid, transcriptome, Illumina, genome sequencing, cDNA synthesis, Nextera XT, Invitrogen
Disclaimer
This assay has been used primarily in aresearch setting and is not necessarily validated from clinical use.
Abstract
The protocol allows rapid RNA metagenome sequencing of pathogens of clinical respiratory samples. The protocol has been designed to be simple and quick to enable the utilization of existing sample extracts and data back within 24h from start to finish (i.e. library prep - sequencing - analysis). The method also uses commonly available reagents that pairs with amplicon based WGS assays, and works by essentially making rRNA depleted ds-cDNA and library amplification with enzyme based tagmentation using Nextera XT DNA Library Preparation KitIllumina, Inc.Catalog #FC-131-1096 .
Materials
Quick-RNA Viral KitZymo ResearchCatalog #R1034
ezDNase™ EnzymeThermo FisherCatalog #11766051
QIAseq FastSelect -rRNA HMR KitQiagenCatalog #334385
QIAseq FastSelect –5S/16S/23S KitQiagenCatalog #335921
SuperScript™ IV VILO™ Master MixInvitrogen - Thermo FisherCatalog #11756050
Sequenase Version 2.0 DNA PolymeraseThermo FisherCatalog #70775Y200UN
Mag-Bind® TotalPure NGSOmega BiotekCatalog #M1378-01
Nextera XT DNA Library Preparation KitIllumina, Inc.Catalog #FC-131-1096
Qubit™ 1X dsDNA High Sensitivity (HS) Assay KitInvitrogen - Thermo FisherCatalog #Q33231
Buffer EBQiagenCatalog #19086
High Sensitivity D1000 ScreenTapeAgilent TechnologiesCatalog #5067-5584 High Sensitivity D1000 ReagentsAgilent TechnologiesCatalog #5067-5585
Protocol materials
Nextera XT DNA Library Preparation KitIllumina, Inc.Catalog #FC-131-1096
Buffer EBQiagenCatalog #19086
Quick-RNA Viral KitZymo ResearchCatalog #R1034
SuperScript™ IV VILO™ Master MixInvitrogen - Thermo FisherCatalog #11756050
Sequenase Version 2.0 DNA PolymeraseThermo FisherCatalog #70775Y200UN
Nextera XT DNA Library Preparation KitIllumina, Inc.Catalog #FC-131-1096
Qubit™ 1X dsDNA High Sensitivity (HS) Assay KitInvitrogen - Thermo FisherCatalog #Q33231
Mag-Bind® TotalPure NGSOmega BiotekCatalog #M1378-01
ezDNase™ EnzymeThermo FisherCatalog #11766051
High Sensitivity D1000 ScreenTapeAgilent TechnologiesCatalog #5067-5584
High Sensitivity D1000 ReagentsAgilent TechnologiesCatalog #5067-5585
QIAseq FastSelect -rRNA HMR KitQiagenCatalog #334385
QIAseq FastSelect –5S/16S/23S KitQiagenCatalog #335921
Quick-RNA Viral KitZymo ResearchCatalog #R1034
Mag-Bind® TotalPure NGSOmega BiotekCatalog #M1378-01
Qubit™ 1X dsDNA High Sensitivity (HS) Assay KitInvitrogen - Thermo FisherCatalog #Q33231
Mag-Bind® TotalPure NGSOmega BiotekCatalog #M1378-01
High Sensitivity D1000 ScreenTapeAgilent TechnologiesCatalog #5067-5584
Buffer EBQiagenCatalog #19086
Buffer EBQiagenCatalog #19086
Qubit™ 1X dsDNA High Sensitivity (HS) Assay KitInvitrogen - Thermo FisherCatalog #Q33231
Safety warnings
Please refer to the Safety Data Sheets (SDS) for health and environmental hazards.
RNA extraction
RNA extraction
Extract RNA with Quick-RNA Viral KitIllumina, Inc.Catalog #R1034 , following manufacturer's instructions.
Note
This protocol can be used to sequence pathogens of clinical respiratory samples, including nose and/or throat swab, nasal discharge or aspirates. For samples collected for the purpose of next generation sequencing (NGS), theQuick-RNA Viral KitZymo ResearchCatalog #R1034 is used to rapidly isolate high quality viral RNA from the source sample.
However we have also demonstrated the usefulness of this method using the residual viral extracts from clinical samples following diagnostic RT-PCRs, which include extracts off both Qiagen BioRobot EZ1 and Roche MagnaPure 96 platforms. Noting that the first step removes genomic DNA (gDNA), so total nucleic acid or purified RNA can be used. Please ensure appropriate PPE is used at all times and relevant containment protocols are followed to avoid unnecessary exposure to potentially infectious samples.
DNase treatment
DNase treatment
Setup the following reaction mix for each sample along with a no template control (NTC):
| A | B | |
| Reagent | Volume (µL) | |
| 10X ezDNase Buffer | 1 | |
| ezDNase enzyme | 1 | |
| DNA/RNA | 8 |
total reaction volume - 10µL per sample
Mix well by gently pipetting up and down, and briefly centrifuge.
Incubate the reaction as follows:
| A | B | |
| Temperature (°C) | Time (mm:ss) | |
| 37 | 10:00 | |
| 4 | ∞ |
After incubation, spin down and place reaction on ice.
10m
rRNA depletion
rRNA depletion
Add the following to the previous reaction (Setup reaction on ice).
| A | B | |
| Reagent | Volume (µL) | |
| previous reaction | 10 | |
| FastSelect Mix | 1 |
Total reaction volume - 11µL per sample; FastSect Mix is a mixture of QIAseq FastSelect -rRNA HMR, QIAseq FastSelect -5S/16S/23S, and water, with the ratio of (QIAseq FastSelect -rRNA HMR):(QIAseq FastSelect -5S/16S/23S):water=1:1:1.
Mix well by pipetting up and down, and briefly centrifuge.
Incubate the reaction as follows:
| A | B | |
| Temperature (°C) | Time (mm:ss) | |
| 75 | 02:00 | |
| 70 | 02:00 | |
| 65 | 02:00 | |
| 60 | 02:00 | |
| 55 | 02:00 | |
| 37 | 02:00 | |
| 25 | 02:00 | |
| 4 | ∞ |
After incubation, spin down and place reaction on ice.
14m
1st strand DNA synthesis
1st strand DNA synthesis
Add the following to the previous reaction (Setup reaction on ice).
| A | B | |
| Reagent | Volume (µL) | |
| previous reaction | 11 | |
| water | 5 | |
| SuperScript IV VILO Master Mix (5X) | 4 |
total reaction volume - 20µL per sample
Mix well by pipetting up and down, and briefly centrifuge.
Incubate the reaction as follows:
| A | B | |
| Temperature (°C) | Time (mm:ss) | |
| 25 | 10:00 | |
| 50 | 20:00 | |
| 85 | 05:00 | |
| 4 | ∞ |
After incubation, spin down and place reaction on ice.
35m
2nd strand DNA synthesis
2nd strand DNA synthesis
Add the following to the previous reaction (Setup reaction on ice).
| A | B | |
| Reagent | Volume (µL) | |
| previous reaction | 20 | |
| water | 11 | |
| Sequenase reaction buffer (5X) | 8 | |
| Sequenase Dilution | 1 |
total reaction volume - 40µL per sample; Sequenase Dilution is a mixture of Sequenase Dilution Buffer and Sequenase Version 2.0 DNA Polymerase, with the ratio of (Sequenase Dilution Buffer):(Sequenase Version 2.0 DNA Polymerase)=2:1.
Mix well by pipetting up and down, and briefly centrifuge.
Incubate the reaction as follows:
| A | B | |
| Temperature (°C) | Time (mm:ss) | |
| 4 | ∞ | |
| 37 | 10:00 | |
| 95 | 02:00 | |
| 4 | ∞ |
Slow ramp (0.1°C/second) from 4°C to 37°C; After incubation, spin down and place reaction on ice.
17m 30s
Add the following to the previous reaction (Setup reaction on ice).
| A | B | |
| Reagent | Volume (µL) | |
| previous reaction | 40 | |
| Sequenase Dilution | 1 |
total reaction volume - 41µL per sample
Mix well by pipetting up and down, and briefly centrifuge.
Incubate the reaction as follows:
| A | B | |
| Temperature (°C) | Time (mm:ss) | |
| 37 | 30:00 | |
| 4 | ∞ |
After incubation, spin down and place reaction on ice.
30m
ds-cDNA purification
ds-cDNA purification
Perform purification of the previous reaction with magnetic beads (Mag-Bind® TotalPure NGSIllumina, Inc.Catalog #M1378-01 ).
Note
0.8X bead reatio (32.8 µL Mag-Bind® TotalPure NGSOmega BiotekCatalog #M1378-01 ).
Resuspend the dried bead pellet with 20 µL Buffer EBQiagenCatalog #19086 .
DNA libarary preparation and sequencing
DNA libarary preparation and sequencing
5m
5m
Note
The protocol follows the Nextera XT DNA Library Preparation KitIllumina, Inc.Catalog #FC-131-1096 except for six changes: i) neat ds-cDNA from previous reaction is used for template, ii) optimised tagment incubation time is used in tagmentation DNA step, iii) IDT® for Illumina Nextera DNA Unique Dual Indexes (Illumina, USA) are used for libraries amplification indexes, iv) optimised cycles are used in libraries amplification step, v) Mag-Bind® TotalPure NGSOmega BiotekCatalog #M1378-01 is used for libraries clean up, and vi) libraries are manually normalised rather than with the provided normalisation beads.
Combine 10 µL Tagment DNA Buffer (TD) and 5 µL neat ds-cDNA from previous reaction for each sample into a PCR plate or strip.
Mix well by pipetting up and down 10 times.
Add 5 µL Amplicon Tagment Mix (ATM) to each well on top of the TD/DNA mix.
Mix well by pipetting up and down 10 times. Seal and briefly centrifuge.
Incubate the reaction mix as follows:
| A | B | |
| Temperature (°C) | Time (mm:ss) | |
| 55 | 07:00 | |
| 10 | ∞ |
Following tagmentation, immediately remove the reaction from the thermocycler.
7m
Add 2.5 µL Neutralize Tagment Buffer (NT) to stop the reaction.
Mix well by pipetting up and down 10 times. Seal and briefly centrifuge.
Incubate at Room temperature for 00:05:00 .
5m
Add indexes and Nextera PCR Master Mix (NPM) to the neutralised tagmentation reaction for each sample as below:
| A | B | |
| Reagent | Volume (µL) | |
| previous reaction | 25 | |
| IDT® for Illumina Nextera DNA Unique Dual Indexes | 10 | |
| Nextera PCR Master Mix (NPM) | 15 |
total reaction volume - 50µL per sample
Mix well by pipetting up and down 10 times. Seal and briefly centrifuge.
Incubate the reaction as follows:
| A | B | C | |
| Temperature (°C) | Time (mm:ss) | cycles | |
| 72 | 03:00 | 1X | |
| 95 | 00:30 | 1X | |
| 95 | 00:10 | 16X | |
| 55 | 00:30 | ||
| 72 | 00:30 | ||
| 72 | 05:00 | 1X | |
| 4 | ∞ |
After incubation, spin down and place reaction on ice.
25m 10s
Perform purification of the previous reaction with magnetic beads (Mag-Bind® TotalPure NGSIllumina, Inc.Catalog #M1378-01 ).
Note
0.8X bead reatio (40 µL Mag-Bind® TotalPure NGSOmega BiotekCatalog #M1378-01 ).
Resuspend the dried bead pellet with 30 µL Buffer EBQiagenCatalog #19086 .
Quantify all purified DNA using
Qubit™ 1X dsDNA High Sensitivity (HS) Assay KitIllumina, Inc.Catalog #Q33231 .
Pool the individual libraries equally in DNA amount based on the Qubit values. Here, we assume the libraries will have similar fragment lengths and distributions.
Quantify the final pool of libraries using
Qubit™ 1X dsDNA High Sensitivity (HS) Assay KitIllumina, Inc.Catalog #Q33231 .
Analyse 2 µL final pool of libraries with High Sensitivity D1000 ScreenTapeIllumina, Inc.Catalog #5067-5584 ensuring the whole fragment peak is captured.
Scale the calculated molarity from the Tapestation to the Qubit DNA concentration using the following formula:
Final molarity = (Tapestation DNA molarity) x (Qubit DNA concentration) / (Tapestation DNA concentration)
Dilute the final pooled libraries down to 1 nanomolar (nM) (at least 50μL) usingBuffer EBIllumina, Inc.Catalog #19086 , and add PhiX sequencing control if required.
Combine 10 µL 1nM libary pool and 90 µL Buffer EBIllumina, Inc.Catalog #19086 to dilute the final pool of libraries to 0.1 nanomolar (nM) for loading.
Load 20 µL 0.1nM library pool into a defrosted Illumina iSeq cartridge with flow cell.
Run in Illumina iSeq.
Equipment
iSeq
NAME
sequencer
TYPE
Illumina
BRAND
ILM20021532
SKU
LINK