TITLE: RAPIDprep: A simple, fast protocol for RNA metagenomic sequencing of clinical samples
AUTHORS: Karan Kim, Racheltulloch, John-Sebastian Eden

[DESCRIPTION]
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   .




[STEPS]
SECTION: RNA extraction
1. Extract RNA with  , following manufacturer's instructions.
 


SECTION: DNase treatment
3. Mix well by gently pipetting up and down, and briefly centrifuge.
SECTION: DNA libarary preparation and sequencing
18. 
 

SECTION: DNA libarary preparation and sequencing
39. Run in Illumina iSeq.
 

SECTION: DNase treatment
2. Setup the following reaction mix for each sample along with a no template control (NTC): 
 



SECTION: rRNA depletion
5. Add the following to the previous reaction (Setup reaction on ice).
 

SECTION: rRNA depletion
6. Mix well by pipetting up and down, and briefly centrifuge.
SECTION: rRNA depletion
7. Incubate the reaction as follows:
 

SECTION: DNase treatment
4.  Incubate the reaction as follows:
 





SECTION: 1st strand DNA synthesis
8. Add the following to the previous reaction (Setup reaction on ice).
 

SECTION: 1st strand DNA synthesis
9. Mix well by pipetting up and down, and briefly centrifuge.
SECTION: 1st strand DNA synthesis
10. Incubate the reaction as follows:
 

SECTION: 2nd strand DNA synthesis
11. Add the following to the previous reaction (Setup reaction on ice).
 

SECTION: 2nd strand DNA synthesis
12. Mix well by pipetting up and down, and briefly centrifuge.
SECTION: 2nd strand DNA synthesis
13. Incubate the reaction as follows:
 


SECTION: 2nd strand DNA synthesis
14. Add the following to the previous reaction (Setup reaction on ice).
 

SECTION: 2nd strand DNA synthesis
15. Mix well by pipetting up and down, and briefly centrifuge.
SECTION: 2nd strand DNA synthesis
16. Incubate the reaction as follows:
 

SECTION: ds-cDNA purification
17. Perform purification of the previous reaction with magnetic beads ( ).
 

SECTION: DNA libarary preparation and sequencing
19. Combine 10 µL and 5 µL for each sample into a PCR plate or strip.
SECTION: DNA libarary preparation and sequencing
20. Mix well by pipetting up and down 10 times.
SECTION: DNA libarary preparation and sequencing
21. Add 5 µL to each well on top of the TD/DNA mix.

SECTION: DNA libarary preparation and sequencing
22. Mix well by pipetting up and down 10 times. Seal and briefly centrifuge.
SECTION: DNA libarary preparation and sequencing
24. Add 2.5 µL to stop the reaction.

SECTION: DNA libarary preparation and sequencing
25. Mix well by pipetting up and down 10 times. Seal and briefly centrifuge.
SECTION: DNA libarary preparation and sequencing
26. Incubate at Room temperature for 5 min.

SECTION: DNA libarary preparation and sequencing
31. Quantify all purified DNA using 
  .
SECTION: DNA libarary preparation and sequencing
32. 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.
SECTION: DNA libarary preparation and sequencing
23. Incubate the reaction mix as follows:
 

SECTION: DNA libarary preparation and sequencing
27. Add indexes and Nextera PCR Master Mix (NPM) to the neutralised tagmentation reaction for each sample as below:
 

SECTION: DNA libarary preparation and sequencing
28. Mix well by pipetting up and down 10 times. Seal and briefly centrifuge.
SECTION: DNA libarary preparation and sequencing
29. Incubate the reaction as follows:
 

SECTION: DNA libarary preparation and sequencing
30. Perform purification of the previous reaction with magnetic beads ( ).
 

SECTION: DNA libarary preparation and sequencing
34. Analyse 2 µL with   ensuring the whole fragment peak is captured.

SECTION: DNA libarary preparation and sequencing
35. 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)
SECTION: DNA libarary preparation and sequencing
36. Dilute the final pooled libraries down to 1 nanomolar (nM) (at least 50μL) using , and add PhiX sequencing control if required.

SECTION: DNA libarary preparation and sequencing
37. Combine 10 µL and 90 µL   to dilute the final pool of libraries to 0.1 nanomolar (nM) for loading.

SECTION: DNA libarary preparation and sequencing
38. Load 20 µL into a defrosted Illumina iSeq cartridge with flow cell.

SECTION: DNA libarary preparation and sequencing
33. Quantify the final pool of libraries using 
  .