Clinical metagenomic sequencing - CSF RNA and DNA Illumina MiSeq

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

A	B
Positive control
Negative control

Table: Sample details and input volume

The following consumables are required for this step:

The following equipment is required for this step:

Equipment

Value	Label
NUCLISENS easyMag	NAME
Nucleic acid extraction	TYPE
bioMerieux	BRAND
N/A	SKU

Equipment

Value	Label
Centrifuge	NAME
Benchtop Centrifuge	TYPE
Eppendorf	BRAND
5405000441	SKU
Any benchtop centrifuge will suffice	SPECIFICATIONS

280x g,4°C

Briefly spin the samples.

Equipment

Value	Label
Centrifuge	NAME
Benchtop Centrifuge	TYPE
Eppendorf	BRAND
5405000441	SKU
Any benchtop centrifuge will suffice	SPECIFICATIONS

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

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

Incubate at room temperature for 0h 10m 0s. Program the sample order on the easyMAG instrument and load the required plastics.

Equipment

Value	Label
NUCLISENS easyMag	NAME
Nucleic acid extraction	TYPE
bioMerieux	BRAND
N/A	SKU

Add 50µL of easyMAG magnetic silica to each lysis buffer tube.

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.

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.

Briefly centrifuge the samples.

280x g,4°C

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

Add 10µL 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.

Incubate at Room temperature for 0h 10m 0s . Program the sample order on the easyMAG instrument and load the required disposable plastics. Set the elution volume to 35µL .

Add 50µL of easyMAG magnetic silica to each sample and internal control containing lysis buffer tube.

Add the contents of the lysis buffer tube to the easyMAG processing wells and start the instrument.

Store 20µL of extracted total nucleic acids in a labelled tube and store at -20°C until ready to proceed to library preparation. The remaining 15µL of extracted total nucleic acids is used generate cDNA from the RNA fraction of extracted total nucleic acids.

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:

Equipment

Value	Label
Centrifuge	NAME
Benchtop Centrifuge	TYPE
Eppendorf	BRAND
5405000441	SKU
Any benchtop centrifuge will suffice	SPECIFICATIONS

Prior to starting:

• Add 96mL of 100% ethanol to the 24mL 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.

Prepare DNAse I master mix as follows:

A	B	C
DNA digestion buffer	5	65
DNAse I enzyme (1 U/ul)	5	65
Nuclease-free water	25	325
Total	35	455

Table: DNAse I master mix

Add 12µL of DNAse/RNAse-Free Water directly to the column matrix and centrifuge.

10000-16000x g

Add 35µL of DNAse I master mix to each sample tube with 15µL of total nucleic acids and mix my inverting gently.

Incubate at Room temperature for 0h 15m 0s . Label a Zymo-Spin IC column and an elution tube for each sample and set aside.

Add 100µL RNA binding buffer to each sample

Add 150µL of 100% ethanol to each sample and mix by inverting

Transfer the sample to its labelled Zymo-Spin IC column in a collection tube and centrifuge. Discard the flow-through.

10000-16000x g

Add 400µL RNA Prep Buffer to each column and centrifuge. Discard the flow-through.

10000-16000x g

Add 700µL RNA Wash Buffer to each column and centrifuge. Discard the flow-through.

10000-16000x g

Add 400µL RNA Wash Buffer to each column and centrifuge. Transfer the column to a labelled elution tube.

10000-16000x g

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:

Equipment

Value	Label
SimpliAmp Thermal Cycler	NAME
PCR	TYPE
Applied Biosystems	BRAND
A24811	SKU
Any standard PCR thermocycler will suffice	SPECIFICATIONS

Prepare priming master mix as follows On ice:

A	B	C
50ng/ul random hexamers	1	13
10mM dNTP mix	1	13
Total	2	26

Table: Priming master mix

Combine the priming master mix with 11µL of purified RNA

Incubate the priming reaction at 65°C for 0h 5m 0s and then put the reaction On ice for 0h 1m 0s. Set the thermocycler lid to 105°C

Prepare the 1st strand synthesis master mix as follows On ice:

A	B	C
5X SSIV Buffer	4	52
100mM DTT	1	13
Ribonuclease inhibitor	1	13
Superscript IV RT (200U/ul)	1	13
Total	7	91

Table: 1st strand master mix

Add the 13µL of RNA with annealed primers to 7µL of 1st strand master mix On ice

Incubate the reaction in a thermocycler with the lid set to 105°C as follows:

A	B
23	10
55	10
80	10
4	Hold

Table: 1st strand thermocycler parameters

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:

Equipment

Value	Label
SimpliAmp Thermal Cycler	NAME
PCR	TYPE
Applied Biosystems	BRAND
A24811	SKU
Any standard PCR thermocycler will suffice	SPECIFICATIONS

Prepare the 2nd strand cDNA synthesis master mix as follows On ice:

A	B	C
Nuclease-free water	55	715
5X second strand reaction mix	20	260
Second strand enzyme mix	5	65
Total	80	1040

Table: 2nd strand synthesis master mix

Add 20µL of 1st strand synthesis product to 80µL of 2nd strand synthesis master mix

Incubate the reaction at 16°C for 1h 0m 0s . Set the thermocycler lid to 40°C .

Stop the reaction by adding 6µL of 0.5Molarity (M) EDTA, 8

Purify the cDNA using beads.

The following consumables are required for this step:

The following equipment is required for this step:

Equipment

Value	Label
Magnetic Stand-96	NAME
ThermoFisher Scientific	BRAND
AM10027	SKU

Prior to starting:

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

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

Incubate for 0h 2m 0s at Room temperature

Place the sample plate on a magnetic stand and allow the solution to clear (0h 5m 0s )

Remove 22µL of the supernatant and transfer to a clean nuclease-free tube

Incubate for 0h 5m 0s at Room temperature

Place the sample plate on a magnetic stand and allow the solution to clear (0h 5m 0s )

Discard the supernatant without disturbing the beads

Add 200µL of 80% ethanol to each well with the sample plate on the magnetic stand and incubate at room temperature for 0h 0m 30s .

Discard the supernatant without disturbing the beads

Repeat the wash steps once. Use a P20 pipette to remove residual ethanol from each well

Air dry the beads (±0h 2m 0s) but do not allow the surface to crack.

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

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:

A	B
Forward	5'-GGATATGTCTAAGTCTGTTGC-3'
Reverse	5'-CAGACAACTCGGGTGCG-3'

Ellis MD, Hoak JM, Ellis BW, Brown JA, Sit TL, Wilkinson CA, Reed TD, Welbaum GE. Quantitative real-time PCR analysis of individual flue-cured tobacco seeds and seedlings reveals seed transmission of tobacco mosaic virus. Phytopathology. 2020 Jan 19;110(1):194-205. The following consumables are required for this step:

The following equipment is required for this step:

Equipment

Value	Label
CFX96 Touch Real-Time PCR	NAME
qPCR	TYPE
Bio-Rad	BRAND
#1855195	SKU

Prepare the master mix as follows On ice

A	B	C
iTaq Universal SYBR reaction mix (2X)	10	130
iScript RT	0.25	3.25
Nuclease-free water	2.75	35.75
Forward primer	1	13
Reverse primer	1	13
Total volume	15	195

Table: Spiked internal control PCR master mix. Sample volume per reaction is 5ul

Add 5µL of post 2nd strand synthesis clean-up product to 15µL of master mix

Run the following PCR program on a qPCR machine reading the SYBR green fluorophore

A	B	C
95	60	1
95	10	40
58	20 (+Read)

Table: Spiked internal control PCR thermocycler parameters. The internal control spike-in should be to a target Ct value of 30±2. The kit reverse transcriptase step is skipped.

Prepare the tagmentation reaction from the kit.

The following consumables are required for this step:

The following equipment is required for this step:

Equipment

Value	Label
SimpliAmp Thermal Cycler	NAME
PCR	TYPE
Applied Biosystems	BRAND
A24811	SKU
Any standard PCR thermocycler will suffice	SPECIFICATIONS

Equipment

Value	Label
Magnetic Stand-96	NAME
ThermoFisher Scientific	BRAND
AM10027	SKU

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

Pool 15µL of extracted total nucleic acids (from initial nucleic acid purification step) and 15µL of purified cDNA (prepared from purified and isolated RNA) in a

Place the plate on the magnetic stand and wait for the solution to clear 0h 3m 0s

Remove and discard the supernatant

Repeat the wash step

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

Prepare the tagmentation master mix as follows:

A	B	C
Bead-linked transposomes (BLT)	11	132
Tagmentation buffer 1 (TB1)	11	132
Total	22	264

Table: Tagmentation master mix. Vortex to mix.

Add 20µL of master mix to 30µL of pooled DNA and mix by pipetting up and down 10 times

Run the following program on a thermocycler with the lid set to 100°C

A	B	C
Tagmentation	55	15
Hold	10	Hold

Table: Tagmentation thermocycler parameters.

Add 10µL of tagmentation stop buffer to each reaction and pipette up and down slowly to resuspend

Run the following program on a thermocycler with the lid set to 100°C

A	B	C
Incubation	37	15
Hold	10	Hold

Table: Tagmentation stop thermocycler parameters.

Place the sample plate on a magnetic stand and allow it to clear 0h 3m 0s

Remove and discard the supernatant

Wash by removing the plate from the magnetic stand and adding 100µL of tagment wash buffer directly onto the beads, pipetting up and down slowly to resuspend

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:

Equipment

Value	Label
SimpliAmp Thermal Cycler	NAME
PCR	TYPE
Applied Biosystems	BRAND
A24811	SKU
Any standard PCR thermocycler will suffice	SPECIFICATIONS

Equipment

Value	Label
Centrifuge	NAME
Benchtop Centrifuge	TYPE
Eppendorf	BRAND
5405000441	SKU
Any benchtop centrifuge will suffice	SPECIFICATIONS

Equipment

Value	Label
Plate centrifuge	NAME
Centrifuge	TYPE
Any	BRAND
N/A	SKU

Equipment

Value	Label
Magnetic Stand-96	NAME
ThermoFisher Scientific	BRAND
AM10027	SKU

Equipment

Value	Label
Qubit Fluorometer	NAME
Fluorometer	TYPE
Invitrogen	BRAND
Q33238	SKU

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

A	B
Positive control
Negative control

Table: Sample identifier and barcoding details.

Prepare amplification master mix as follows:

A	B	C
Enhanced PCR mix (EPM)	22	264
Nuclease-free water	22	264
Total	44	528

Table: Amplification master mix.

Vortex the sample purification beads and add 81µL to each well containing supernatant and pipette 10 times to mix

Incubate the plate for 0h 5m 0s at Room temperature

Place plate on a magnetic stand and wait for the liquid to clear 0h 5m 0s

Remove and discard the supernatant

Wash by adding 200µL of freshly prepared 80% ethanol to each well without mixing

Incubate for 0h 0m 30s

Remove and discard the supernatant

Repeat the wash step once

Use a small volume pipettor remove residual ethanol

Air-dry for up to 0h 1m 0s but do not let the beads crack

Vortex and centrifuge 280x g

Add 32µL of resuspension buffer (RSB) to the beads and resuspend

Incubate at room temperature for 0h 2m 0s

Place on a magnetic stand and allow the liquid to clear (0h 2m 0s )

Transfer 30µL of supernatant to a new plate

Quantify the libraries using a Qubit fluorometer using:

A	B
Positive control
Negative control

Table: Sample identifier and library concentration after clean-up 1.

With the plate/tube from the post-tagmentation clean-up, discard the supernatant and remove from the magnetic stand

Add 40µL 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 280x g

Add adaptors to each sample – 5µL i7 adaptor and 5µL i5 adaptor and record the used combination – and mix by pipetting up and down 10 times and centrifuge 280x g

Run the following PCR program with the lid set to 100°C

A	B	C
68	180	1
98	180	1
98	45	12
62	30
68	120
68	60	1
10	Hold	Hold

Table: Amplification thermocycler parameters. The cycle number can be adjusted based on input DNA.

Centrifuge the samples 280x g

Place the plate on a magnetic stand and allow the liquid to clear0h 5m 0s

Transfer 45µL supernatant from each well into a new plate

Additional clean-up: Libraries quantified as >10ng/ul will undergo a double-sided clean-up

Add 25µL of cleaned library to a new plate (double-sided clean-up plate 1)

Add 60µL of nuclease-free water

Add 45µL of SPB

Incubate at Room temperature for 0h 5m 0s

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 15µL of undiluted SPB to each well to be used.

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

Additional clean-up: Libraries quantified as <10ng/ul will undergo a single-sided clean-up

Add 25µL of libary to a new plate (corresponding wells in the double-sided clean-up plate 2 from may be used)

Add 60µL of nuclease-free water

Add 60µL of SPB

Finalisation of libraries

Incubate the plate(s) (double-sided clean-up plate 2 ) at Room temperature for 0h 5m 0s

Add 30µL of resuspension buffer (RSB) to the beads and resuspend

Incubate at room temperature for 0h 2m 0s

Place the plate on a magnetic stand and wait for the liquid to clear (0h 2m 0s )

Transfer 28µL of supernatant to a nuclease-free tube

Place on a magnetic stand and wait for the liquid to clear (0h 2m 0s )

Remove and discard the supernatant

Wash by adding 200µL of freshly prepared 80% ethanol to each well without mixing

Incubate for 0h 0m 30s

Remove and discard the supernatant

Repeat the wash step once

Using a small volume pipettor remove residual ethanol

Air-dry for up to 0h 1m 0s but do not let the beads crack

Quantification and QC of libraries

Quantify the libraries using a Qubit fluorometer using:

A	B
Positive control
Negative control

Table: Sample identifier and library concentration after final clean-up.

(Optionally) evaluate the library mean fragment size using a TapeScreen instrument. If this is not done, use a default mean fragment size of 600bp.

Calculate the Molar concentration of the library using the following formula:

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:

Equipment

Value	Label
MiSeq	NAME
Sequencer	TYPE
illumina	BRAND
SY-410-1003	SKU

Equipment

Value	Label
Centrifuge	NAME
Benchtop Centrifuge	TYPE
Eppendorf	BRAND
5405000441	SKU
Any benchtop centrifuge will suffice	SPECIFICATIONS

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 1h 0m 0s 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 0.2Molar 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)

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

Start the sequencer

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

A	B
4nM pooled libraries	5
0.2M NaOH	5
Total volume	10

Table: Library denaturation.

Vortex briefly and spin-down the denaturing libraries and incubate for 0h 5m 0s at Room temperature

Stop the reaction by adding 990µL of chilled HT-1 buffer

Invert several times and spin-down the solution

Repeat the denaturation and stopping of a 4nanomolar (nM) PhiX library

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

Further dilute the library (with added PhiX) to a 10picomolar (pM) concentration and keep the reaction On ice until the library is loaded into the MiSeq reagent cartridge

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

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.

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).

Install conda, instructions are available at:

https://docs.conda.io/projects/conda/en/latest/user-guide/install/linux.html

To install the required packages run the following commands:

#Install biobakery  (Linux Ubuntu)
conda install -c biobakery biobakery_workflows 

#Install bio bakery dependency (Linux  Ubuntu)
conda install tbb=2020.2

#Biobakery database (Linux Ubuntu)
kneaddata_database --download human_genome bowtie2 $PATH_TO_WORKING_DIRECTORY
```The kneaddata database can also be directly dowloaded from:

[http://huttenhower.sph.harvard.edu/kneadData_databases](http://huttenhower.sph.harvard.edu/kneadData_databases)

#Install kraken2 (Linux Ubuntu) conda install -c bioconda kraken2

#Install spades (Linux Ubuntu) conda install -c bioconda spades

#Install DIAMOND (Linux Ubuntu) conda install -c bioconda diamond

#Install krakentools (Linux Ubuntu) conda install -c bioconda krakentools

#Install PEAR (Linux Ubuntu) conda install -c bioconda pear

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.

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:

#Move trimmomatic to bin (Linux Ubuntu)
sudo mv $PATH_AFTER_DOWNLOAD /bin

Additional dependencies:

Download the {Reviewed (Swiss-Prot)} fasta file to generate the DIAMOND database from:

https://www.uniprot.org/help/downloads

Analyse the raw sequencing reads as follows:

1. Human read, contaminant and low-complexity read removal with kneaddata:

#Run kneaddata (Linux Ubuntu)
kneaddata --input $PAIRED_END_RAW_READ_1 --input $PAIRED_END_RAW_READ_2 --reference-db Homo_sapiens_hg37_and_human_contamination_Bowtie2_v0.1 --output ~/$WORKING_DIRECTORY/ --threads 30 --trimmomatic /bin/Trimmomatic-0.39/
```Review the kneaddata paired reads in FastQC and make any required modifications to the trimming algorithm if necessary.



2) Taxonomic classification using kraken2: 

#Run kraken2 (Linux Ubuntu) kraken2 --db NAME_OF_REPORT_WITH_DBNAME_DATACLEANSTRATEGY_DATE_SAMPLE --paired KNEADDATA_PAIRED_READ_2 --output $KRAKENFILE

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:

#Extract unclassified reads (Linux Ubuntu) extract_kraken_reads.py -k KNEADDATA_PAIRED_READ_1 -s2 UNMATCHED_FORWARD -o2 $UNMATCHED_REVERSE

4) Generate contigs from unclassified reads using SPAdes: 

#SPAdes (Linux Ubuntu) spades.py --meta -1 UNMATCHED_REVERSE -o $SPADES_OUTPUT_DIRECTORY

5) Generate merged paired-end reads if no contigs are generated:

#Assemble paired-end reads (Linux Ubuntu) pear -f UNMATCHED_REVERSE -o $OUTPUT_DIRECTORY

#Generate DIAMOND database (Linux Ubuntu) diamond makedb --in DIAMOND_DATABASE_NAME

#Run DIAMOND (Linux Ubuntu) diamond blastx -d CONTIGS_OR_ASSEMBLED_READS.fasta -o $DATABASE_MATCHES.tsv

Data visualisation can be done using Pavian. Installation and usage instructions are available here:

https://github.com/fbreitwieser/pavian

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 | A | B | | --- | --- | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | |

A: Sample ID, B: Sample volume