Complete genome assembly of Hawai’i environmental nontuberculous mycobacteria reveals unexpected co-isolation with methylobacteria

Nontuberculous mycobacteria (NTM) are ubiquitous environmental opportunistic pathogens that can cause chronic lung disease. Within the United States, Hawai’i has the highest incidence of NTM lung disease, though the precise reasons are yet to be fully elucidated. One possibility is the high prevalence of NTM in the Hawai’i environment acting as a potential reservoir for opportunistic NTM infections. Through our previous initiatives to collect and characterize NTM in Hawai’i, community scientists of Hawai’i have collected thousands of environmental samples for sequencing. Here, these community scientists were invited for the first time into a high school lab in O’ahu for a genomic sequencing workshop, where participants sequenced four of the collected isolate genomic samples using the Oxford Nanopore Technologies MinION sequencer. Participants generated high quality long read data that when combined with short read Illumina data yielded complete bacterial genomic assemblies suitable for in-depth analysis. The gene annotation analysis identified a suite of genes that might help NTM thrive in the Hawai’i environment. Further, we found evidence of co-occurring methylobacteria, revealed from the sequencing data, suggesting that in some cases methylobacteria and NTM may coexist in the same niche, challenging previously accepted paradigms. The sequencing efforts presented here generated novel insights regarding the potential survival strategies and microbial interactions of NTM in the geographic hot spot of Hawai’i. We highlight the contributions of community scientists and present an activity that can be reimplemented as a workshop or classroom activity by other research groups to engage their local communities.


Instructor Materials Learning Objectives
• Reflect on the potential implications of environmental NTM being present in the local environment.
• Demonstrate safe and effective laboratory techniques.
• Describe MinION sequences and how it can be used to answer research questions.
• Be able to identify sequence mutations and predict the functional implications on the protein.

Safety Issues
Student protocols were designed to avoid contact with any harmful reagents or biological components.

Master Supply List
A list of supplies and catalogue numbers is provided below.The curriculum was designed to use only equipment found in a typical microbial teaching laboratory and materials available from Oxford Nanopore Technologies (ONT).In addition, minimal computational resources are required.○ The MinION device, Computer, and MinKNOW software can be substituted for the MinION Mk1C.This is an all-in-one device that controls the sequencing run and displays results.• iPads for streaming (optional)

Post-Sequencing Analysis
• Basecaller ○ There are several options for basecalling and each has different system requirements.The MinKNOW software (run from a computer or the MinION Mk1C) offers a fast-basecalling option for immediate results.External Guppy software offers high-accuracy basecalling for improved results.Guppy can be run on CPUs but this can be very slow so a NVIDIA GPU-based system is recommended.Compute resources may also be accessed through Amazon Web Services (AWS), Google Cloud Platform (GCP), EPI2ME, or other cloud-based platforms.

• Assembler
○ Many assemblers are available.For long-read only assembly, we recommend Canu.If Illumina data is also available, we recommend hybrid assembly with Unicycler.

Bioinformatics Tutorial
• Computers for students

Bioinformatics Tutorial
There are many different types of analysis that can be performed after DNA sequencing.The purpose of this tutorial is to introduce students to gene identification, sequence alignment, and protein visualization.

MinION Sequencing Lab Protocol Overview
Rapid Barcoding Kit1

Protocol step What's actually happening
Testing the pore activity of the flow cell using the MinKNOW software.
The MinION detects which nanopores on the flow cell are available by sequencing DNA test fragments that are present in the storage buffer.The minimum number of available pores recommended is 800.
Barcoding the DNA in each library separately Each piece of genomic DNA within a library is enzymatically cleaved and a pre-assigned barcode is attached to each end of every resulting fragment.
transposase + barcodes genomic DNA Pooling the samples Each barcoded library is then combined in equal proportions to be processed and sequenced together on a single flow cell.

Attaching the sequencing adapters
An enzyme attaches sequencing adapters to each end of every barcoded DNA fragment.A single motor protein comes pre-attached to each sequencing adapter.

Priming the flow cell
The flow cell must be primed to receive the final DNA sequencing library.This step removes the storage buffer and provides ATP for the motor proteins.
Preparing the final DNA sequencing library The barcoded, pooled and adapted DNA is then mixed with loading beads, which help to distribute the DNA fragments evenly across the sensor array during loading.
Loading the DNA library The final library is loaded directly onto the sensor array for sequencing.

Teacher's Prep Guide
Required equipment and reagents: • Micropipettes and tips

MinION Sequencing Library Preparation Protocol Rapid Barcoding Kit 1
Step Protocol Action What's actually happening Testing pore activity of the flow cell 1 Connect the MinION to the computer and install the flow cell.
Using the MinKNOW software, run a pore test experiment.
The MinION detects which pores are actively sequencing using DNA test fragments in the storage buffer.
Fragment the genomic DNA and attach the barcodes 2 In a small PCR tube, prepare the following recipe for 3 Using a thermocycler, incubate the reaction at the following temperatures:

°C HOLD
The transposome is activated by raising the reaction temperature to 30 °C.The enzyme is then deactivated at 80 °C, and the product is preserved by holding the temperature at 4 °C.

ʻĀina-Informatics
Pool the barcoded samples 4 In a 1.5 mL Eppendorf tube, combine equal volumes of each library for a total volume of 10 µL.
Running multiple libraries together on a single flow cell, or multiplexing, makes the most efficient use of the flow cell.

Attach the sequencing adapters 5
To your DNA, add 1 µL of Rapid Adapter (RAP).
Mix gently by flicking the tube, and spin down.
Incubate the reaction at room temperature for 5 min.
Each sequencing adapter comes pre-attached with a single motor protein.An enzyme attaches an adapter to each end of every barcoded fragment of DNA in your reaction.

Key Terms
Library: A sequencing library consists of all the DNA fragments originating from a single sample that will be sequenced simultaneously.A "pooled" or "multiplexed" library consists of all the DNA fragments originating from multiple source libraries that are combined in order to be sequenced simultaneously.
Barcode: A sequence of DNA manufactured to be unique enough that, when attached to a subset of DNA fragments within a pooled library, can be used to algorithmically sort the resulting sequences into their original source libraries.
Multiplexing: Combining multiple libraries to be sequenced simultaneously on a single flow cell.
Sequencing adapter: A sequence of synthetic DNA which attaches to each barcoded DNA fragment and allows the fragment to interface with the nanopore.
Motor protein: A protein attached to each sequencing adapter, and therefore to each DNA fragment to be sequenced, which ratchets one strand of the DNA fragment into the nanopore one base at a time.

Loading the MinION Flow Cell Protocol 1
Step Protocol Action What's actually happening Prime the MinION flow cell 1 Prepare the Priming Mix by adding 30 µL of Flush Tether (FLT) directly into 1 tube of Flush Buffer (FB).
Mix by vortexing.
This Priming Mix contains ATP which powers the motor protein.
2 Open the priming port.
Set a P-1000 pipette to 200 µL and insert the tip into the priming port.Using the pipette wheel, draw back a small amount of storage buffer into the pipette tip.
This step is to remove any air, which is damaging to the nanopores in the sensor array, from the priming port.
3 Slowly load 800 µL of the Priming Mix in through the priming port, avoiding the introduction of air bubbles.Wait 5 min.
The Priming Mix forces the yellow storage buffer off the sensor array and into the waste channel.
Prepare the library for loading onto the flow cell 4 In a 1.5 mL Eppendorf tube, prepare the library for loading according to the following recipe:

Reagent Volume
Sequencing The Loading Beads settle quickly and must be resuspended immediately before use.The beads help to distribute the DNA fragments evenly across the sensor array during the loading process.
1 Adapted for use with Oxford Nanopore Technology's Flow Cell Priming Kit (EXP-FLP002), which is included with the Rapid Sequencing Kit (SQK-RAD004) and the Rapid Barcoding Kit (SQK-RBK004).

ʻĀina-Informatics
E. Tong, ʻĀina-Informatics, ʻIolani School.Rev 9.2021 Complete the final step in priming the flow cell 5 Gently lift the SpotON sample port cover.
Slowly load 200 µL of the Priming Mix into the priming port, pausing for buffer coming up from the SpotON port to recede before loading more.
This step ensures that when the library is loaded, it is continuous with the Priming Mix, and therefore no air is introduced into the flow cell.
Load the DNA library 6 Mix the prepared library by gently pipetting up and down to resuspend the loading beads.
Add 75 µL of your prepared library in a drop-wise fashion into the SpotON sample port, ensuring that each drop flows into the port before adding the next.
Return the SpotON sample port and priming port covers to their closed positions.
Your sample is now in contact with the nanopores in the sensor array and can be sequenced.

Start the sequencing run 7
In the MinKNOW software, begin the sequencing run by providing a run ID, sample ID, library prep kit information and a location for data storage.
Select fast basecalling and barcoding options if applicable.
The software will begin detecting the electrical signals generated by bases passing through each active pore and send the data to the cloud for basecalling.

Parts of a MinION Flow Cell
Washing the MinION Flow Cell Protocol 1 Step Protocol Action What's actually happening Prepare the flow cell wash mix 1 If sequencing is still ongoing, note the number of active pores in MinKNOW and stop data acquisition.
In a 1.5 mL Eppendorf tube, prepare the Flow Cell Wash Mix according to the following recipe, mix by pipetting and store on ice until needed.

Reagent Volume
Wash This step should only remove fluid from the waste channel, not from the sensor array.
Flush the previous libraries from the flow cell sensor array 3 Open the priming port.Set a P-1000 pipette to 200 µL and insert the tip into the priming port.Using the pipette wheel, draw back a small amount of fluid into the pipette tip.
This step is to remove any air, which is damaging to the nanopores in the sensor array, from the priming port.
4 Slowly load 400 µL of the Flow Cell Wash Mix in through the priming port, avoiding the introduction of air bubbles.Close the priming port and wait 60 min.
The Flow Cell Wash Mix digests up to 99.9% of the DNA from the previous run.
1 Adapted for use with Oxford Nanopore Technology's Flow Cell Wash Kit (EXP-WSH004).

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Remove waste from the flow cell waste channel 5 With both the priming port and SpotOn port closed, use a P-1000 pipette to remove all the fluid in the waste channel from waste port 1.
This step again removes fluid from the waste channel, not from the sensor array.
To reuse the flow cell right away 6 To prepare the flow cell for short term storage, proceed to step 7.

Use the Priming and Loading the MinION Flow Cell
Protocol to re-prime the flow cell for immediate reuse.
The washed flow cell can be reused immediately.
To prepare the flow cell for short term storage 7 Open the priming port.Set a P-1000 pipette to 200 µL and insert the tip into the priming port.Using the pipette wheel, draw back a small amount of fluid into the pipette tip.
This step is to remove any air, which is damaging to the nanopores in the sensor array, from the priming port.
8 Add 500 µL room temperature Storage Buffer (S) in through the priming port, avoiding the introduction of bubbles.Close the priming port.
The Storage Buffer preserves the sensor array and allows for subsequent pore checks.
Remove remaining waste from flow cell and store 9 Use a P-1000 pipette to remove all the fluid in the waste channel from waste port 1.
Once all waste is removed, store the flow cell at 4 °C.

Overview
This lesson asks you to compare gene sequences between one wild-type and one of a variety of mutant nontuberculous mycobacterial strains.You will identify mutations as single-nucleotide polymorphisms (SNPs) and then make an inference on whether your variant strain will impact the activity of the protein or antibiotics that target the protein.

Objectives
By the end of this activity you will be able to: Based on this information, which location warrants further investigation, by whole genome sequencing?

Part 3
You are interested in variations in protein sequences of these strains as well, corresponding to the RNA polymerase beta subunit.Translate the environmental isolate sequences to amino acid sequences, using GeneMarkS.

Part 4
Perform a Multiple Sequence Alignment on the translated protein sequences, to examine amino acid variation of the rpoB protein, in the environmental NTM, using CLUSTAL.

Part 5
In addition to being an essential protein involve in RNA transcription, the RNA polymerase beta protein is an important drug target, in both NTM and M. tuberculosis.
Search the Protein Data Bank (PDB), using the Environmental Isolate 5 sequence, to see if protein structures with similar sequences have been determined.

2
With both the priming port and SpotOn port closed, use a P-1000 pipette to remove all the fluid in the waste channel from waste port 1.