TITLE: Clinical metagenomic sequencing - CSF RNA and DNA  Illumina MiSeq
AUTHORS: Gert Marais, Ziyaad Valley-Omar, Kathleen Bateman, Kate McMullen, Dieter van der Westhuizen, Moepeng Maseko, Suzaan Marais, Diana Hardie, Adrian Brink

[DESCRIPTION]
This protocol was developed to evaluate unbiased cerebrospinal fluid metagenomics at a referral centre in South Africa. Commercial kit protocols are generally used unmodified and limited use of in-house developed methods or reagents is made. This maintains the simplicity and accessibility of the protocol which can serve as a base for evaluation of process improvements. 

[GUIDELINES]
Please note: This protocol is intended for research use only. 

1) This protocol functions optimally with low cell-count CSF specimens with minimal human DNA. High cell-count specimens should ideally be evaluated using sequencing protocols that include general microbiome enrichment or targeted enrichment and/or human DNA depletion. 

2) The number of individuals performing the clinical metagenomic sequencing (CMS) protocol and the number of days over which it is performed determines the order of steps. 

3) Sodium hypochlorite is the preferred cleaning solution 

4) The preferred run size is 10 samples and 2 controls though the cellularity of used CSF may alter the preferred run size due to increased quantity of human DNA. Master mix volumes are calculated and presented here for this run size. 

5) The total hands-on time (including incubation and thermocycler time) for the protocol is 14 hours and 11 minutes. This assumes a single operator and 10 samples with 2 controls. The time estimate excludes sequencing time (generally 48-56 hours with the MiSeq v3 600-cycle kits) and bioinformatics time. It is generally advisable to perform the protocol over 2 days.

6) Prior to use samples should be stored at -70 °C and defrosted immediately prior to use. 


[STEPS]
SECTION: Nucleic Acid Purification
147. Perform total nucleic acid extraction using the NUCLISENS easyMAG platform or a similar total nucleic acid extraction platform. 
Process all samples using appropriate infectious prevention and control protocols relevant to the included samples 
CSF sample input volume of 200-1000ul is acceptable for the easyMAG platform but may differ for other platforms 
Add 10ul of internal control into each sample: The specific internal control used is laboratory dependent and the specific volume will depend on the prior characteristics and prior optimisation of this internal control
Set the easyMAG elution volume to 35ul 
A positive control is included in each run: 200-1000ul of the NIBSC Viral Multiplex Reference 11/242
A negative control is included in each run: Lysis buffer only with added internal control
Label the lysis buffer tubes (easyMAG Lysis buffer tubes) and elution tubes (Eppendorf LoBind 1.5ml tubes) prior to starting the extraction procedure
Do not use on board lysis 
10 samples and 2 controls is the preferred run size 
 

SECTION: Nucleic Acid Purification
0.2. Remove CSF samples from-70 °C storage and defrost on ice . Label the sample elution tubes (1 for RNA and 1 for total nucleic acids for each sample) and easyMAG lysis buffer tubes while the samples defrost.

SECTION: Nucleic Acid Purification
0.147. 280 x g, 30 s, 4 °C 
Briefly spin the samples.
 

SECTION: Nucleic Acid Purification
0.147. Add 200-1000 µL of CSF sample to an easyMAG lysis buffer tube. CAUTION: Prevent cross-contamination between samples. 

SECTION: Nucleic Acid Purification
0.147. Add 10 µL Internal control to each sample in lysis buffer. CAUTION: Prevent cross-contamination between samples. 

SECTION: Nucleic Acid Purification
0.147. Incubate at room temperature for 10 min. Program the sample order on the easyMAG instrument and load the required plastics.
 


SECTION: Nucleic Acid Purification
0.147. Add 50 µL of easyMAG magnetic silica to each lysis buffer tube.

SECTION: Nucleic Acid Purification
0.147. Add the contents of the lysis buffer tubes with added sample, internal control and magnetic silica to the easyMAG processing wells and start the instrument. 
SECTION: Nucleic Acid Purification
0.147. 
SECTION: Nucleic Acid Purification
0.1. 
 

Perform total nucleic acid extraction using the NUCLISENS easyMAG platform or another similar total nucleic acid extraction platform. 
The preferred run size is 10 samples and 2 controls 
The preferred positive control is the NIBSC Viral Multiplex Reference 11/242
The preferred negative control is the extraction platform lysis buffer
The internal control used is laboratory specific and the volume used is dependent on its specific characteristics and lab optimisation prior to use
Enter the sample details in the table below


 

The following consumables are required for this step:

 
  

The following equipment is required for this step:


 

 

SECTION: Nucleic Acid Purification
0.3. Briefly centrifuge the samples.

280 x g, 30 s, 4 °C 


SECTION: Nucleic Acid Purification
0.4. Add 200-1000 µL of sample to a easyMAG lysis buffer tube. Similarly add 200-1000 µL of the positive control to a lysis buffer tube. CAUTION: Avoid sample cross-contamination.

  

SECTION: Nucleic Acid Purification
0.5. Add 10 µL of internal control to each sample in lysis buffer (including the positive control and negative control lysis buffer tube which contains no sample). CAUTION: Avoid sample cross-contamination.

SECTION: Nucleic Acid Purification
0.6. Incubate at Room temperature for 10 min . Program the sample order on the easyMAG instrument and load the required disposable plastics. Set the elution volume to 35 µL .



SECTION: Nucleic Acid Purification
0.7. Add 50 µL of easyMAG magnetic silica to each sample and internal control containing lysis buffer tube.

SECTION: Nucleic Acid Purification
0.8. Add the contents of the lysis buffer tube to the easyMAG processing wells and start the instrument.
SECTION: Nucleic Acid Purification
0.9. Store 20 µL of extracted total nucleic acids in a labelled tube and store at -20 °C until ready to proceed to library preparation. The remaining 15 µL of extracted total nucleic acids is used generate cDNA from the RNA fraction of extracted total nucleic acids. 

SECTION: cDNA from purified RNA
1. Perform RNA purification from a fraction of the extracted total nucleic acids using the  

The following consumables are required for this step:

  
  

The following equipment is required for this step:
 

Prior to starting:

Add 96 mL of 100% ethanol to the 24 mL RNA wash buffer concentrate. 
Reconstitute the lyophilised DNAse I with nuclease-free water to a concentration of 1 U/ul or defrost an aliquot of previously reconstituted DNAse I.
SECTION: cDNA from purified RNA
1.1. Prepare DNAse I master mix as follows:

 

SECTION: cDNA from purified RNA
1.2. Add 35 µL of DNAse I master mix to each sample tube with 15 µL of total nucleic acids and mix my inverting gently.

SECTION: cDNA from purified RNA
1.3. Incubate at Room temperature for 15 min . Label a Zymo-Spin IC column and an elution tube for each sample and set aside.  
SECTION: cDNA from purified RNA
1.4. Add 100 µL RNA binding buffer to each sample

SECTION: cDNA from purified RNA
1.5. Add 150 µL of 100% ethanol to each sample and mix by inverting

SECTION: cDNA from purified RNA
1.6. Transfer the sample to its labelled Zymo-Spin IC column in a collection tube and centrifuge. Discard the flow-through.

10000-16000 x g, 30 s 

SECTION: cDNA from purified RNA
1.7. Add 400 µL RNA Prep Buffer to each column and centrifuge. Discard the flow-through. 

10000-16000 x g, 30 s 

SECTION: cDNA from purified RNA
1.8. Add 700 µL RNA Wash Buffer to each column and centrifuge. Discard the flow-through. 

10000-16000 x g, 30 s 

SECTION: cDNA from purified RNA
1.9. Add 400 µL RNA Wash Buffer to each column and centrifuge. Transfer the column to a labelled elution tube. 

10000-16000 x g, 1 min 
SECTION: cDNA from purified RNA
1.10. Add 12 µL of DNAse/RNAse-Free Water directly to the column matrix and centrifuge. 

10000-16000 x g, 1 min 

SECTION: cDNA from purified RNA
2. Perform first-strand synthesis on the purified RNA using the  kit.

The following consumables are required for this step:

  

The following equipment is required for this step:
 

SECTION: cDNA from purified RNA
2.1. Prepare priming master mix as follows on ice:
 

SECTION: cDNA from purified RNA
2.2. Combine the priming master mix with 11 µL of purified RNA 

SECTION: cDNA from purified RNA
2.3. Incubate the priming reaction at 65 °C for 5 min and then put the reaction on ice for 1 min. Set the thermocycler lid to 105 °C 

SECTION: cDNA from purified RNA
2.4. 
 
 Prepare the 1st strand synthesis master mix as follows on ice:
 

 
 
SECTION: cDNA from purified RNA
2.5. Add the 13 µL of RNA with annealed primers to 7 µL of 1st strand master mix on ice

SECTION: cDNA from purified RNA
2.6. Incubate the reaction in a thermocycler with the lid set to 105 °C  as follows:
 

SECTION: cDNA from purified RNA
3. Perform 2nd strand synthesis on the 1st strand synthesis product using the   kit.

The following consumables are required for this step:

  

The following equipment is required for this step:
 

SECTION: cDNA from purified RNA
3.1. Prepare the 2nd strand cDNA synthesis master mix as follows on ice:
 
 
 

 
 
SECTION: cDNA from purified RNA
3.2. Add 20 µL of 1st strand synthesis product to 80 µL of 2nd strand synthesis master mix 

SECTION: cDNA from purified RNA
3.3. Incubate the reaction at 16 °C for 60 min . Set the thermocycler lid to 40 °C .

SECTION: cDNA from purified RNA
3.4. Stop the reaction by adding 6 µL of 0.5 Molarity (M) EDTA, pH 8 

SECTION: cDNA from purified RNA
4. Purify the cDNA using  beads. 

The following consumables are required for this step:


  
  
  

The following equipment is required for this step:
 
Prior to starting: 

Prepare 80% ethanol 
Label the purified cDNA tubes
Bring the   beads to Room temperature and resuspend by vortexing.

SECTION: cDNA from purified RNA
4.1. Add 180 µL (1.8X) of   to 100 µL of 2nd strand synthesis product from 4.4 and mix well by pipetting up and down 10 times. 

SECTION: cDNA from purified RNA
4.2. Incubate for 5 min at Room temperature 


SECTION: cDNA from purified RNA
4.3. Place the sample plate on a magnetic stand and allow the solution to clear (5 min )

SECTION: cDNA from purified RNA
4.4. Discard the supernatant without disturbing the beads
SECTION: cDNA from purified RNA
4.5. Add 200 µL of 80% ethanol to each well with the sample plate on the magnetic stand and incubate at room temperature for 30 s .

SECTION: cDNA from purified RNA
4.6. Discard the supernatant without disturbing the beads
SECTION: cDNA from purified RNA
4.7. Repeat the wash steps once. Use a P20 pipette to remove residual ethanol from each well
SECTION: cDNA from purified RNA
4.8. Air dry the beads (±2 min) but do not allow the surface to crack.

SECTION: cDNA from purified RNA
4.9. Remove the plate from the magnetic rack. Elute the DNA from the beads by adding 25 µL nuclease-free water to the beads. Mix well by pipetting up and down 10 times. 

SECTION: cDNA from purified RNA
4.10. Incubate for 2 min at Room temperature 

SECTION: cDNA from purified RNA
4.11. Place the sample plate on a magnetic stand and allow the solution to clear (5 min )
SECTION: cDNA from purified RNA
4.12. Remove 22 µL of the supernatant and transfer to a clean nuclease-free tube

SECTION: Pre-Sequencing QC PCR
5. Perform a QC real-time PCR using the   kit. The primers should be specific to the internal control used. This protocol used Tobacco Mild Green Mosaic virus as an internal control with the following primers diluted as per the iTaq kit package insert: 

 
The following consumables are required for this step:

  

The following equipment is required for this step: 
 

SECTION: Pre-Sequencing QC PCR
5.1. Prepare the master mix as follows on ice 

 

SECTION: Pre-Sequencing QC PCR
5.2. Add 5 µL of post 2nd strand synthesis clean-up product to 15 µL  of master mix

SECTION: Pre-Sequencing QC PCR
5.3. Run the following PCR program on a qPCR machine reading the SYBR green fluorophore
 

SECTION: Library Preparation
6. 
Prepare the tagmentation reaction from the  kit.

The following consumables are required for this step: 

  

The following equipment is required for this step:
 

 

Prior to starting:
Bring the bead-linked transposomes (BLT), tagmentation buffer 1 (TB1), tagmentation stop buffer (TSB) and tangent wash buffer (TWB) to Room temperature  and vortex to mix

SECTION: Library Preparation
6.1. Pool  15 µL of extracted total nucleic acids (from initial nucleic acid purification step) and 15 µL  of purified cDNA (prepared from purified and isolated RNA) in a  

SECTION: Library Preparation
6.2. Prepare the tagmentation master mix as follows:

 

SECTION: Library Preparation
6.3. Add 20 µL  of master mix to 30 µL of pooled DNA and mix by pipetting up and down 10 times

SECTION: Library Preparation
6.4. Run the following program on a thermocycler with the lid set to 100 °C 

 

SECTION: Library Preparation
6.5. Add 10 µL of tagmentation stop buffer to each reaction and pipette up and down slowly to resuspend

SECTION: Library Preparation
6.6. Run the following program on a thermocycler with the lid set to 100 °C 
 
 

 
 
SECTION: Library Preparation
6.7. Place the sample plate on a magnetic stand and allow it to clear 3 min 

SECTION: Library Preparation
6.8. Remove and discard the supernatant 
SECTION: Library Preparation
6.9. Wash by removing the plate from the magnetic stand and adding 100 µL  of tagment wash buffer directly onto the beads, pipetting up and down slowly to resuspend

SECTION: Library Preparation
6.10. Place the plate on the magnetic stand and wait for the solution to clear 3 min 

SECTION: Library Preparation
6.11. Remove and discard the supernatant 
SECTION: Library Preparation
6.12. Repeat the wash step

SECTION: Library Preparation
6.13. Remove the plate from the magnetic stand and add 100 µL of tagment wash buffer directly onto the beads and resuspend. Cover the plate and place on the magnetic stand until the amplification step.

SECTION: Library Preparation
7. Amplify the tagmented DNA using the   and the  kit. 

The following consumables are required for this step and steps immediately following: 

 
  
  
  

The following equipment is required for this step:
 

 

 

 

 

Prior to starting this step and and steps immediately following:
Thaw and bring to Room temperature  the index adaptors. Thaw the Enhanced PCR mix on ice .
Bring the sample purification beads (SPB) to Room temperature 
Thaw the resuspension buffer (RSB) and allow it to come to Room temperature 
Prepare 80% ethanol


 



SECTION: Library Preparation
7.1. Prepare amplification master mix as follows:


 

SECTION: Library Preparation
7.2. Vortex and centrifuge 280 x g, 10 s 

SECTION: Library Preparation
7.3. With the plate/tube from the post-tagmentation clean-up, discard the supernatant and remove from the magnetic stand
SECTION: Library Preparation
7.4. Add 40 µL  of PCR master mix directly onto the beads of each sample and pipette up and down to ensure the beads are fully suspended before sealing the plate and centrifuge  280 x g, 10 s  

SECTION: Library Preparation
7.5. Add adaptors to each sample – 5 µL  i7 adaptor and 5 µL  i5 adaptor and record the used combination – and mix by pipetting up and down 10 times and centrifuge  280 x g, 10 s  

SECTION: Library Preparation
7.6. Run the following PCR program with the lid set to 100 °C 

 

SECTION: Library Preparation
7.7. Centrifuge the samples 280 x g, 1 min 

SECTION: Library Preparation
7.8. Place the plate on a magnetic stand and allow the liquid to clear5 min 

SECTION: Library Preparation
7.9. Transfer 45 µL  supernatant from each well into a new plate

SECTION: Library Preparation
7.10. Vortex the sample purification beads and add 81 µL to each well containing supernatant and pipette 10 times to mix

SECTION: Library Preparation
7.11. Incubate the plate for 5 min at Room temperature 

SECTION: Library Preparation
7.12. Place plate on a magnetic stand and wait for the liquid to clear 5 min 

SECTION: Library Preparation
7.13. Remove and discard the supernatant
SECTION: Library Preparation
7.14. Wash by adding 200 µL  of freshly prepared 80% ethanol to each well without mixing
SECTION: Library Preparation
7.15. Incubate for 30 s 

SECTION: Library Preparation
7.16. Remove and discard the supernatant
SECTION: Library Preparation
7.17. Repeat the wash step once
SECTION: Library Preparation
7.18. Use a small volume pipettor remove residual ethanol
SECTION: Library Preparation
7.19. Air-dry for up to 1 min but do not let the beads crack

SECTION: Library Preparation
7.20. Add 32 µL of resuspension buffer (RSB) to the beads and resuspend

SECTION: Library Preparation
7.21. Incubate at room temperature for 2 min 

SECTION: Library Preparation
7.22. Place on a magnetic stand and allow the liquid to clear (2 min )

SECTION: Library Preparation
7.23. Transfer 30 µL of supernatant to a new plate 

SECTION: Library Preparation
7.24. Quantify the libraries using a Qubit fluorometer using:
  

 

SECTION: Library Preparation
8. Additional clean-up: Libraries quantified as >10ng/ul will undergo a double-sided clean-up

SECTION: Library Preparation
8.1. Add 25 µL of cleaned library to a new plate (double-sided clean-up plate 1)

SECTION: Library Preparation
8.2. Add 60 µL of nuclease-free water 

SECTION: Library Preparation
8.3. Add 45 µL of SPB 

SECTION: Library Preparation
8.4. Incubate at Room temperature for 5 min 

SECTION: Library Preparation
8.5. Place the plate on a magnetic stand and allow the liquid to clear. During this time, to a second plate (double-sided clean-up plate 2) add 15 µL of undiluted SPB to each well to be used. 

SECTION: Library Preparation
8.6. Transfer 125 µL of supernatant from double-sided clean-up plate 1 to the predispensed 15 µL of SPB in double-sided clean-up plate 2. Discard double-sided clean-up plate 1.

SECTION: Library Preparation
9. Additional clean-up: Libraries quantified as <10ng/ul will undergo a single-sided clean-up
SECTION: Library Preparation
9.1. Add 25 µL of libary to a new plate (corresponding wells in the double-sided clean-up plate 2 from may be used)

SECTION: Library Preparation
9.2. Add 60 µL of nuclease-free water 

SECTION: Library Preparation
9.3. Add 60 µL of SPB 

SECTION: Library Preparation
10. Finalisation of libraries
SECTION: Library Preparation
10.1. Incubate the plate(s) (double-sided clean-up plate 2 ) at Room temperature for 5 min 

SECTION: Library Preparation
10.2. Place on a magnetic stand and wait for the liquid to clear (2 min )

SECTION: Library Preparation
10.3. Remove and discard the supernatant
SECTION: Library Preparation
10.4. Wash by adding 200 µL  of freshly prepared 80% ethanol to each well without mixing
SECTION: Library Preparation
10.5. Incubate for 30 s 

SECTION: Library Preparation
10.6. Remove and discard the supernatant
SECTION: Library Preparation
10.7. Repeat the wash step once
SECTION: Library Preparation
10.8. Using a small volume pipettor remove residual ethanol
SECTION: Library Preparation
10.9. Air-dry for up to 1 min but do not let the beads crack
SECTION: Library Preparation
10.10. Add 30 µL of resuspension buffer (RSB) to the beads and resuspend
SECTION: Library Preparation
10.11. Incubate at room temperature for 2 min 

SECTION: Library Preparation
10.12. Place the plate on a magnetic stand and wait for the liquid to clear (2 min )

SECTION: Library Preparation
10.13. Transfer 28 µL of supernatant to a nuclease-free tube

SECTION: Library Preparation
11. Quantification and QC of libraries
SECTION: Library Preparation
11.1. Quantify the libraries using a Qubit fluorometer using:
  

 

SECTION: Library Preparation
11.2. (Optionally) evaluate the library mean fragment size using a TapeScreen instrument. If this is not done, use a default mean fragment size of 600bp.
SECTION: Library Preparation
11.3. Calculate the Molar concentration of the library using the following formula: 

   

SECTION: Sequencing
12. Prepare the quantified and quality controlled libraries for loading and load the Sequencing instrument. 

The following consumables are required for this step:

  
  
  
  
  

The following equipment is required for this step:
 

 
Prior to starting this step and and steps immediately following:
Defrost the PhiX control
Ensure the MiSeq instrument doesn't require maintenance or a weekly wash
Remove the Illumina Miseq v3 reagent cartridge from -20 °C storage and defrost in a water bath approximately 60 min before its intended use. Alternatively place it at 4 °C overnight prior to its intended use
Defrost buffer HT-1 on ice  and keep at 4 °C until use
Prepare a fresh solution of 0.2 Molar NaOH 
Create the sample sheet using the appropriate software that will be used for the MiSeq sequencing run (this will indicate the number of cycles, the library preparation parameters and the sample names and indexes for use by the instrument to set the run parameters and demultiplex the output data)

SECTION: Sequencing
12.1. Dilute the prepared libraries to 4 nanomolar (nM) using nuclease-free water and pool them in a single LoBind Eppendorf tube

SECTION: Sequencing
12.2. Combine the diluted library and  0.2 Molar NaOH solution in a LoBind Eppendorf tube to denature the libraries as follows: 

 


SECTION: Sequencing
12.3. Vortex briefly and spin-down the denaturing libraries and incubate for 5 min at Room temperature 

SECTION: Sequencing
12.4. Stop the reaction by adding 990 µL of chilled HT-1 buffer

SECTION: Sequencing
12.5. Invert several times and spin-down the solution
SECTION: Sequencing
12.6. Repeat the denaturation and stopping of a 4 nanomolar (nM) PhiX library

SECTION: Sequencing
12.7. The denatured library concentration is 20 picomolar (pM) . Remove 100 µL  from the library and add 100 µL of the 20 picomolar (pM)  denatured and stopped PhiX library 

SECTION: Sequencing
12.8. Further dilute the library (with added PhiX) to a 10 picomolar (pM) concentration and keep the reaction on ice until the library is loaded into the MiSeq reagent cartridge

SECTION: Sequencing
12.9. Follow the prompts on the MiSeq screen to load the flow cell, wash buffer and reagent cartridge and load 600 µL of 10 picomolar (pM) pooled libary solution (with PhiX) prior to loading the cartridge.

SECTION: Sequencing
12.10. Start the sequencer
SECTION: Bioinformatics
14. The analysis described below was performed on a computer with a 32-core Intel(R) Xeon(R) CPU and 124Gb of RAM. Analysis was done locally and the computer was running Linux (Ubuntu distribution).
SECTION: Bioinformatics
14.1. Install conda, instructions are available at: 
https://docs.conda.io/projects/conda/en/latest/user-guide/install/linux.html
SECTION: Bioinformatics
14.2. To install the required packages run the following commands: 
 

 

 
The kneaddata database can also be directly dowloaded from:
http://huttenhower.sph.harvard.edu/kneadData_databases
 

 

 

 

 

SECTION: Bioinformatics
14.3. Additional dependencies: 

Kraken2 databases can be constructed de novo or are available as pre-constructed databases at: 
https://benlangmead.github.io/aws-indexes/k2

Download the standard database and the EuPathDB48 database and extract into the working directory where you will analyse the sequencing data.
SECTION: Bioinformatics
14.4. Additional dependencies: 

Download the Trimmomatic (Trimmomatic-0.39) binary file from: 
usadellab.org/cms/?page=trimmomatic 

and move it (after extraction) to bin as follows:
 

SECTION: Bioinformatics
14.6. Analyse the raw sequencing reads as follows:

1) Human read, contaminant and low-complexity read removal with kneaddata:
 
Review the kneaddata paired reads in FastQC and make any required modifications to the trimming algorithm if necessary.

2) Taxonomic classification using kraken2: 
 

This command is run once for each kraken2 database used. Thus, for this analysis the command twice for the standard and EuPathDB48 databases.

3) Extract unclassified reads using krakentools:
 
The reads not classified by kraken2 are extracted for further analysis. 

4) Generate contigs from unclassified reads using SPAdes: 
 

5) Generate merged paired-end reads if no contigs are generated:
 
6) Generate the DIAMOND database as follows: 
 
 7) Run DIAMOND on generated contigs and/or assembled unclassified reads (if no contigs are generated):
 

SECTION: Bioinformatics
14.5. Additional dependencies: 

Download the {Reviewed (Swiss-Prot)} fasta file to generate the DIAMOND database from: 
https://www.uniprot.org/help/downloads
SECTION: Bioinformatics
14.7. Data visualisation can be done using Pavian. Installation and usage instructions are available here: 
https://github.com/fbreitwieser/pavian
SECTION: Bioinformatics
13. The bioinformatics tools needed to perform the complete analysis are available at: https://github.com/RuanMarais/UCT_metagenomics. Alternatively, basic analysis can be performed as described below.