Characterization of the Archaeome, Bacteriome and Eukaryome in Nasopharyngeal Swabs

DNA was isolated from nasal swabs with amies medium, using approximately 1000µL .

For the Isolation, the QIAmp DNA-Mini Kit by Qiagen was used, following the QiAamp tissue protocol from the ‘QIAamp DNA Mini and Blood Mini Handbook 05/2016’

Centrifugation of sample at 7500rpm until pellet formation.

Replace QIAamp Mini spin column with a new 2 ml collection tube. Discard tube with the filtrate. Centrifugation: 14000rpm

Placing QIAamp Mini spin column in a new 1.5 ml microcentrifuge tube. Discard tube with the filtrate.

Add 100µL. Incubation at room temperature (0h 5m 0s), centrifugation:8000rpm

Repeat Step 1.11: Add the flowthrough of the previous step to the Mini spin column and incubation at room temperature for 0h 5m 0s, centrifugation 8000rpmat .

Concentration measurement with nanophotometer or qubit.

Resuspension of pellet in 180µL

Adding 20µL, vortexing,

Incubate at 56°C until complete lysis. Occasionally vortexing.

Brief Centrifugation of the sample.

Adding 200µL, pulse-vortex afterwards 0h 0m 15s, Incubation at 70°C for 0h 10m 0s . Brief centrifugation of the sample.

Adding 200µL, and mix by pulse-vortexing for 0h 0m 15s. Afterwards, short centrifugation of the sample.

Transfer mixture (including precipitate) to the QIAamp Mini spin column. CAVE: without wetting the rim. Centrifugation: 8000rpm

Replace the QIAamp Mini spin column, use a clean 2 ml collection tube, Discard tube with the filtrate.

Adding 500µL CAVE: without wetting the rim.

Centrifuge:8000rpm.

Replace the QIAamp Mini spin column, use a clean 2 ml collection tube, Discard tube with the filtrate.

Add 500µL to the QIAamp Mini spin column without wetting the rim. Closing of the column, Centrifugation: 14000rpm

PCR

Archaea: Nested PCR

Eukaryotes: single PCR

Primer selection for archaea and eukaryotes

A	B	C	D
Nr.	Name	Primer Name	Sequence (5’  3’)
1	344F	S-D-Arch-0344-fw	5’-acggggygcagcaggcgcga-3’
2	1041R	S-D-Arch-1041-rev	5’-ggccatgcaccwcctctc-3’
3	519F	Arch-519F-Tag	5’-tttctgttggtgctgatattgccagcmgccgcggtaa-3’
4	786R	Arch-786R-Tag	5’-acttgcctgtcgctctatcctcggactacvsgggtatctaat-3’
5	563F	Euk-563F-Tag	5’-tttctgttggtgctgatattgcgccagcavcygcggtaay-3’
6	1132R	Euk-1132R-Tag	5’-acttgcctgtcgctctatcttcccgtcaatthcttyaart-3’

1st PCR Mix:

8µL

12.5µL

2µL

2.5µL

PCR-Run. 1

Primer pair Arch-344-F-1041R / Eck.563F-1132Rtag

Heated Lid: 110 C

Denaturation 95°C 0h 3m 0s

Cycles (30):

Denaturation 95°C 0h 0m 30s

Annealing 55°C 0h 0m 30s

Elongation 72°C``0h 0m 30s

End Cycle

Final Elongation 65°C``0h 5m 0s

2nd PCR (archaea only) Mix:

9.5µL

12.5µL

2µL

1µL

PCR-Run. 2 (nested)

Primer pair Arch-519-F-786Rtag

Heated Lid: 110 C

Denaturation 95°C 0h 3m 0s

Cycles (28):

Denaturation 95°C 0h 0m 30s

Annealing 55°C 0h 0m 30s

Elongation 72°C``0h 0m 30s

End Cycle

Final Elongation 65°C``0h 5m 0s

Gel Electrophoresis

Check, if the wanted sequences were amplified. Ether through classic gel electrophoresis or through microcapillary gel electrophoresis.

Library Preparation + Sequencing:

- 1st Purification

- PCR preparation

- 2nd Purification

- Concentration measurement

1st Purification:

Add 36µL and apply an external magnetic field for 0h 5m 0s. Afterwards discard fluid supernatant.

Add another 150µL . Afterwards discard the fluid supernatant and dry tube with open lid

Resuspend pellet in 15µL

Concentration measurement with nanophotometer

Library preparation:

We used a modified version of the PCR barcoding (96) genomic DNA (SQK-LSK109) protocol by Nanopore.

Quantify the barcoded library using a nanophotometer and pool all barcoded libraries in the desired ratios in a 1.5 ml DNA LoBind Eppendorf tube.

Prepare 1µg in 47µL.

DNA repair and end-prep

Thaw DNA CS (DCS) at RT, spin down, mix by pipetting, and place on ice.

Prepare the NEBNext FFPE DNA Repair Mix and NEBNext Ultra II End repair / dA-tailing Module reagents in accordance with manufacturer’s instructions, and place on ice.

In a 0.2 ml thin-walled PCR tube, mix the following:

1µL 47µL 3.5µL

2µL 3.5µL 3µL

Mix gently by flicking the tube, and spin down.

Using a thermal cycler, incubate at 20°C for 0h 5m 0s and 65°C for 0h 5m 0s

Add 150µL and discard fluid supernatant.

AMPure XP bead clean-up

Resuspend the AMPure XP beads by vortexing.

Transfer the DNA sample to a clean 1.5 ml Eppendorf DNA LoBind tube.

Add 60µL to the end-prep reaction and mix by flicking the tube.

Incubate on a Hula mixer (rotator mixer) for 0h 5m 0s at room temperature.

Prepare 500µL in Nuclease-free water.

Spin down the sample and pellet on a magnet until eluate is clear and colourless.

Keep the tube on the magnet, and pipette off the supernatant.

Keep the tube on the magnet and wash the beads with200µL without disturbing the pellet. Remove the ethanol using a pipette and discard.

Repeat the previous step.

Spin down and place the tube back on the magnet.

Pipette off any residual ethanol. Allow to dry for 0h 0m 30s, but do not dry the pellet to the point of cracking.

Remove the tube from the magnetic rack and resuspend the pellet in 61µL

Incubate for 0h 2m 0s at RT.

Add another 150µL . Afterwards discard the fluid supernatant and dry tube with open lid

Pellet the beads on a magnet until the eluate is clear and colourless.

Remove and retain 61µL into a clean 1.5 ml Eppendorf DNA LoBind tube.

Take forward the repaired and end-prepped DNA into the adapter ligation step.

However, at this point it is also possible to store the sample at 4°C overnight.

Adapter ligation and clean-up (PCR barcoding (96) genomic DNA (SQK-LSK109) protocol by Nanopore)

Although the recommended 3rd party ligase is supplied with its own buffer, the ligation efficiency of Adapter Mix (AMX) is higher when using Ligation Buffer supplied within the Ligation Sequencing Kit.

Spin down the Adapter Mix (AMX) and Quick T4 Ligase, and place on ice.

Thaw Ligation Buffer (LNB) at RT, spin down and mix by pipetting.

Due to viscosity, vortexing this buffer is ineffective. Place on ice immediately after thawing and mixing.

Thaw the Elution Buffer (EB) at RT, mix by vortexing, spin down and place on ice.

To retain DNA fragments of < 3 KB, thaw one tube of Short Fragment Buffer (SFB) at RT, mix by vortexing, spin down and place on ice.

In a 1.5 ml Eppendorf DNA LoBind tube, mix in the following order:

60µL 25µL 10µL

5µL

Mix gently by flicking the tube, and spin down.

Incubate the reaction for 0h 10m 0s at RT. If you have omitted the AMPure purification step after DNA repair and end-prep, do not incubate the reaction for longer than 0h 10m 0s.

Resuspend pellet in 15µL

Resuspend the AMPure XP beads by vortexing. Add 40µL to the reaction and mix by flicking the tube.

Incubate on a Hula mixer (rotator mixer) for 0h 5m 0sat RT.

Spin down the sample and pellet on a magnet. Keep the tube on the magnet, and pipette off the supernatant.

Wash the beads by adding 250µL. Flick the beads to resuspend, spin down, then return the tube to the magnetic rack and allow the beads to pellet. Remove the supernatant using a pipette and discard.

Repeat the previous step.

Spin down and place the tube back on the magnet. Pipette off any residual supernatant. Allow to dry for 0h 0m 30sbut do not dry the pellet to the point of cracking.

Remove the tube from the magnetic rack and resuspend the pellet in 15µL. Spin down and incubate for 0h 10m 0s at RT.

Pellet the beads on a magnet until the eluate is clear and colourless.

Remove and retain 15µL into a clean 1.5 ml Eppendorf DNA LoBind tube.

Quantify 1µL using a Qubit fluorometer. The prepared library is used for loading into the flow cell. Store the library on ice until ready to load.

PCR-preparation: Mix

12.5µL

2µL

9.5µL

1µL

The prepared library is used for loading into the flow cell. Store the library on ice until ready to load.

Priming and loading the SpotON flow cell

Thaw the Sequencing Buffer (SQB), Loading Beads (LB), Flush Tether (FLT) and one tube of Flush Buffer (FB) at RT.

Mix the Sequencing Buffer (SQB), Flush Tether (FLT) and Flush Buffer (FB) tubes by vortexing and spin down at RT.

Open the MinION Mk1B lid and slide the flow cell under the clip. Slide the priming port cover clockwise to open the priming port.

Take care when drawing back buffer from the flow cell. Do not remove more than 20-30 μl, and make sure that the array of pores are covered by buffer at all times. Introducing air bubbles into the array can irreversibly damage pores.

After opening the priming port, check for a small air bubble under the cover. Draw back a small volume to remove any bubbles (a few μl):

Set a P1000 pipette to 200 μl

Insert the tip into the priming port

Turn the wheel until the dial shows 220-230 μl, or until you can see a small volume of buffer entering the pipette tip

To prepare the flow cell priming mix, add 30µL directly to the tube of thawed and mixed Flush Buffer (FB), and mix by vortexing at RT.

Load 800µL mix into the flow cell via the priming port, avoiding the introduction of air bubbles. Wait for 0h 5m 0s. During this time, prepare the library for loading by following the steps below.

Thoroughly mix the contents of the Loading Beads (LB) by pipetting because it contains a suspension of beads which settle very quickly. It is vital that they are mixed immediately before use!

In a new tube, prepare the library for loading as follows: 37.5µL 25.5µL , mixed immediately before use

12µL

Heated Lid: 110 C

Denaturation 95°C``0h 3m 0s

Cycles (18):

Denaturation 95°C``0h 0m 15s

Annealing 62°C``0h 0m 15s

Elongation 65°C``0h 0m 45s

End Cycle

Final Elongation 65°C``0h 5m 0s

Complete the flow cell priming through Gently lifting the SpotON sample port cover to make the SpotON sample port accessible. Load 200µL into the flow cell via the priming port (not the SpotON sample port), avoiding the introduction of air bubbles.

Mix the prepared library gently by pipetting up and down just prior to loading.

Add 75µL to the flow cell via the SpotON sample port in a dropwise fashion. Ensure each drop flows into the port before adding the next.

Gently replace the SpotON sample port cover, making sure the bung enters the SpotON port, close the priming port and replace the lid.

If you using a MinION Mk1C turn basecalling while sequencing on.

Ending the experiment

After your sequencing experiment is complete, if you would like to reuse the flow cell, please follow the Wash Kit instructions and store the washed flow cell at 2-8°C, OR

Follow the returns procedure by washing out the flow cell ready to send back to Oxford Nanopore.

2nd Purification

Add 36µL and apply an external magnetic field for 0h 5m 0s. Afterwards discard fluid supernatant

Add 150µL and discard fluid supernatant.

If you were unable to basecall in real time, perform the basecalling now using the Guppy basecaller (newest version).

https://community.nanoporetech.com/docs/prepare/library_prep_protocols/Guppy-protocol/v/gpb_2003_v1_revag_14dec2018/guppy-software-overview

Now, using the resulting .fastq files, run the WIMP workflow from the Epi2Me software.

(https://nanoporetech.com/resource-centre/epi2me-wimp-workflow-quantitative-real-time-species-identification-metagenomic)

If the graphical output from the WIMP workflow is not sufficient for your analysis, you can download the results in a .csv dataset. Due to the size of this dataset, further analyses may be performed by creating an SQL database.

The data contains the

• filename of the .fastq file
• Read ID --> is the unique primary key, wich enables to identify the read and therefore the sequence
• Run ID
• exit_status (of the read from the WIMP workflow)
• barcode
• taxID (every phylogenetic rank of each species has its own ID, with these IDs the lineage is composed
• name (of the organism)
• score
• lineage

Python scripts

While working on this project, a few Python scripts may be useful, depending on analysis you want to perform.

This script we used to split large files into smaller ones:

#IMPORTANT: this script must be started from the same file directory as your input file!

filecounter=0
filelinecounter=0

inputfilename="file_i_want_to_split.txt"  #set the correct name of the file, you want to #split. 
filename=inputfilename.split(".")[0]
file_lines= open(inputfilename, 'r').readlines()
print(len(file_lines))
while filelinecounter	outputfilename=filename+"_"+str(filecounter).zfill(3)+".txt"  #set #the correct ending for your file here
	print(outputfilename)

	while filelinecounter		outfile.write(file_lines[filelinecounter])
		filelinecounter=filelinecounter+1
	else: filecounter=filecounter+1

This script was used, to append the lenght of each analysed read (or with small changes the whole sequence) to the .csv table:

#IMPORTANT: this script must be started from the same file directory as your input file! 
# This script, the .fastq files from the run you want to analyse and the WIMP.csv file must be in the same directory!


inputfilename="WIMP_inputfile.csv" #change the inputfile here

import os
from multiprocessing import Pool
import concurrent.futures #imports the multithreading library
import shutil
from pathlib import Path
filecounter=0
filelinecounter=0
i=1

# Define a function for the thread
def search_fasta(WIMP_inputline):
	WIMP_inputline=WIMP_inputline.rstrip()
	fastqfilename=WIMP_inputline.split("-",2)[0]+".fastq"
	#print(str(fastqfilename))
	readID=WIMP_inputline.split(",",3)[1]
	#print(str(readID))
	fqfile=open(fastqfilename, 'r').readlines()
	#print("fqfile is open")
	#print(str(fqfile[0]))
	fqcounter=0
	found= False 
	while found == False:
		fqreadID= fqfile[fqcounter*4].split()[0][1:37]
		#print(str(fqreadID))
		if (readID == fqreadID):
			readlenght=len(fqfile[fqcounter*4+1])  # if you want to get the sequence instead of the lenght, remove the len() function.
			#print(str(readlenght))
			found=True
		else: fqcounter=fqcounter+1
	completeline=WIMP_inputline+","+str(readlenght)+"\n"
	#print("Thread")
	return completeline

if __name__ == "__main__":
	dirname = os.path.join("C:/WIMPlenght_tmp")
	os.mkdir(dirname)

	filename=inputfilename.split(".")[0]
	print(filename)
	file_lines= open(inputfilename, 'r').readlines()
	print(len(file_lines))
	while filelinecounter		throughputfilename=filename+"_"+str(filecounter).zfill(6)+".csv"
		print(throughputfilename)
		while filelinecounter			outfile=open(dirname+"/"+throughputfilename, 'a')
			print(filelinecounter)
			outfile.write(file_lines[filelinecounter])
			filelinecounter=filelinecounter+1
			#print(filelinecounter)
		else: 
			filecounter=filecounter+1
			print("Filenumber: ", filecounter)

	print("tmpfiles complete")
	outputfilename=inputfilename.split(".")[0]+"_Output_WIMP&Seqlenght.csv"
	print(outputfilename)

	while i < filecounter:

		tmpfilename=filename+"_"+str(i).zfill(6)+".csv"
		WIMP_lines = open(os.path.join(dirname+"/"+tmpfilename), 'r').readlines() #opens the tmp WIMP outputfile and creates a list with each line as one item in the list
		p=Pool()
		with open(outputfilename, 'a') as outfile:
			result=p.map(search_fasta, WIMP_lines)
			p.close()
			p.join()
			#print(result)
			for f in result:
				#print(f)
				outfile.write(f)
		i=i+1
		print(i)
	else:
		print("task complete")
		shutil.rmtree(dirname)
		print("tmpfiles deleted")