High-quality genome assembly of a <i>Pestalotiopsis</i> fungus using DIY-friendly methods

Joshua L. McGinnis; Daniel J. Giguere

doi:10.12688/f1000research.110351.1

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Brief Report

High-quality genome assembly of a Pestalotiopsis fungus using DIY-friendly methods

[version 1; peer review: 3 approved with reservations]

Joshua L. McGinnis ¹, Daniel J. Giguere ^2,3

PUBLISHED 20 Apr 2022

Author details Author details

¹ EverymanBio, Los Angeles, CA, 90047, USA
² Department of Biochemistry, University of Western Ontario, London, ON, Canada
³ Flow Genomics, Toronto, ON, Canada

Joshua L. McGinnis
Roles: Conceptualization, Data Curation, Formal Analysis, Funding Acquisition, Investigation, Methodology, Project Administration, Resources, Software, Supervision, Validation, Visualization, Writing – Original Draft Preparation, Writing – Review & Editing

Daniel J. Giguere
Roles: Conceptualization, Data Curation, Formal Analysis, Funding Acquisition, Investigation, Methodology, Project Administration, Resources, Software, Supervision, Validation, Visualization, Writing – Original Draft Preparation, Writing – Review & Editing

OPEN PEER REVIEW

REVIEWER STATUS

This article is included in the Bioinformatics gateway.

This article is included in the Genomics and Genetics gateway.

Abstract

Of the millions of fungal species estimated to exist, about 100,000 have been identified, and only approximately 3000 of those have representative genome assemblies available. Here, we isolated a wild species of Pestalotiopsis from the Los Angeles area, extracted DNA in a low-cost environment (e.g., home lab), and generated a high-quality genome assembly using the low-cost Oxford Nanopore MinION sequencing platform. We found that Pestalotiopsis has a genome composed of 7 nuclear chromosomes, comprising 47.7 megabases. Using this genome, we perform a multi-locus phylogenetic analysis and finally, we discuss how this project (costing $300) demonstrates the increased accessibility of whole genome sequencing.

Keywords

Pestalotiopsis, genome assembly, nanopore sequencing, diy

Corresponding authors: Joshua L. McGinnis, Daniel J. Giguere

Competing interests: Joshua McGinnis founded EverymanBio, Daniel Giguere founded Flow Genomics

Grant information: DJG: Mitacs IT24796, Flow Genomics
The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Copyright: © 2022 McGinnis JL and Giguere DJ. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

How to cite: McGinnis JL and Giguere DJ. High-quality genome assembly of a Pestalotiopsis fungus using DIY-friendly methods [version 1; peer review: 3 approved with reservations]. F1000Research 2022, 11:442 (https://doi.org/10.12688/f1000research.110351.1) First published: 20 Apr 2022, 11:442 (https://doi.org/10.12688/f1000research.110351.1) Latest published: 20 Apr 2022, 11:442 (https://doi.org/10.12688/f1000research.110351.1)

Introduction

Accessibility to genome sequencing has typically been limited to large, well-funded institutions due to large capital requirements of DNA sequencing platforms, like many high-throughput Illumina platforms that cost hundreds of thousands of dollars to obtain. However, the recently developed MinION nanopore sequencing platform by Oxford Nanopore Technologies provides both the hardware and reagents needed for genome sequencing for $1000 USD, reducing the financial barrier to DNA sequencing. Other equipment like thermocyclers are also becoming more portable and affordable,¹ reducing the barrier for “do-it-yourself” (DIY) biologists to perform genetic analysis and engineering projects.² The distribution of resources and knowledge between DIY biologists and institutional researchers can advance the rate of development to research and provide lower-cost approaches.³

A significant portion of the approximately 22,000 bioactive microbial metabolites are of fungal origin.⁴ Furthermore, of the estimated three million species of fungi, only about 100,000 species have been identified.⁵ This poses an opportunity to discover and characterize new metabolite-producing fungi that may lead to the commercialization of novel pharmaceuticals, pigments, enzymes and food-production platforms. Pestalotiopsis, the focus of this study, is a diverse genus of Ascomycete fungi, often pathogenic to plants⁶ and commonly isolated as endophytes.⁷ Pestalotiopsis is known to possess potentially valuable metabolic capabilities, including the production of bioactive secondary metabolites like taxol⁸ and the ability to biodegrade polyurethane.⁹

The purpose of this work is 1) to better understand the genome of Pestalotiopsis and 2) to demonstrate that high-quality eukaryotic genome sequencing is now accessible in a DIY environment. To do this, we built a high-quality genome of Pestalotiopsis using nanopore sequencing, highlighting the increased accessibility and affordability of genome sequencing for amateur biologists and early-stage startups.

Methods

Strain and culture conditions

A wild-type strain of Pestalotiopsis was isolated from a wild-type clone of a Laetiporus sp. found growing on an aged hardwood stump at Whiting Ranch Wilderness Park, Orange County, California, United States.

The isolate was maintained on Petri dishes (eBay) containing Malt Extract Agar (MEA) (Amazon.com) media at room temperature in natural indoor ambient light. Microscopic images are shown in Figure 1.

Figure 1. Microscopic images of Pestalotiopsis used in this study.

A. Conidiomata B. Conidia C. Mature colony on MEA.

DNA extraction

A sterile toothpick was used to aseptically transfer a small swath (2-4 mm) of fresh, non-sporulating Pestalotiopsis mycelium from the surface of solid MEA culture into a 0.2 ml microcentrifuge tube containing 30 $μ$ l of 0.5 M NaOH (Home Science Tools, SKU: UN1823, MT) lysing buffer.¹⁰

A makeshift pestle (Figure 2) was crafted from a 200 $μ$ l long pipette tip and quickly melted with the flame from a lighter followed by immediate application to a flat surface sanitized with 70% isopropyl alcohol, in order to form a small flat non-porous tip roughly 1 mm in size. Using the makeshift pestle, the tissue was crushed and macerated in the tube for one to three minutes, or until the tissue was homogeneously suspended in the lysis buffer and no clumps were observed.

Figure 2. Collection of do-it-yourself (DIY) equipment used in this project.

A. DIY Magnetic Bead Separator Rack constructed with neodymium magnets; B. Vibrating Back Massager as DIY Vortex Mixer; C. Makeshift Pestle generated by melting a pipette tip with over a flame and applying the melted tip to a sterile solid surface before hardening into a flat blunt-end.

Figure 3. Estimated molecular weight of DNA.

Sample 1 (5 $μ$ l), 1 kb ladder, Sample 1 (10 $μ$ l), 1 kb ladder, Sample 2 (5 $μ$ l), 1 kb ladder, Sample 2 (10 $μ$ l).

The tube was allowed to sit for 10 min at room temperature. 150 $μ$ l of 100 mM Tris, Boric acid, EDTA (TBE) pH 8.3 buffer (Bioland Scientific, TBE01, CA) was added and the tube was incubated in a thermocycler heat block for 10 minutes at 95 °C. The tissue debris was then pelleted for five minutes at 5000 × g (10,000 rpm) leaving the genomic DNA used as the template for the PCR reaction left remaining in the supernatant.

PCR reaction

The PCR reaction was prepared in a separate 0.2 ml microcentrifuge tube by mixing 9 $μ$ l of sterile distilled water, 0.4 $μ$ l of 10 $μ$ M forward primer, 0.4 $μ$ l of 10 $μ$ M reverse primer, and 11 $μ$ l of 2× Taq PCR Premix (Bioland Scientific, TP01-00, CA). Template DNA was added to the mix by drawing off 1 $μ$ l of supernatant from the top of the tube used in the previous lysis step.

Common fungal DNA barcoding primers ITS1-F (5′-CTTGGTCATTTAGAGGAAGTAA-3′)¹¹ and ITS4 (5′-TCCTCCGCTTATTGATATGC-3′)¹² were used to amplify the internal transcribed spacer (ITS) region of the fungal ribosomal encoding genes. The cycling conditions were as follows: an initial denaturation step (2 min at 94 °C), 35 cycles of denaturation (94 °C for 30 secs), annealing (55 °C for 2 min) and elongation (72 °C for 1 min), and a final elongation step (72 °C for 10 min).

Successful PCR amplification of the barcoding locus was confirmed via electrophoresis using the MiniPCR blueGel system (Amplyus, MA) operating at a fixed 48V.

A total of 5 $μ$ l of PCR product was mixed with 1 $μ$ l of 6× DNA loading buffer (Bioland Scientific, DSB01-01, CA) and loaded into a 1% TBE gel. The gel was run for approximately 30 minutes until a bright band was visible and a size of 400-700 bp approximated by comparison with a 50 bp DNA ladder (Bioland Scientific, DM02-02, CA).

Sequencing

A total of 15 $μ$ l of PCR product was transferred to a new 0.2 ml microcentrifuge, tightly wrapped in parafilm and sent through USPS Priority mail in a small padded envelop for forward-read Sanger sequencing (MCLab, Oakland, CA).

The resulting chromatogram was analyzed, trimmed and error-corrected by hand using SnapGene Viewer (GSL Biotech LLC, CA). The resulting sequence was then searched against NCBI GenBank using BLAST and aligned against type and ex-type sequences.

High molecular weight genomic extraction

Fungal tissue preparation

A double-layer media plate was prepared using a 9 mm Petri dish with a 30 ml Malt Extract Agar (MEA) bottom-layer and a 20 ml Potato Dextrose broth top-layer. The top liquid layer was aseptically inoculated with a sterile toothpick by transferring a small amount of conidia from a sporulating culture into the center of the broth and was gently agitated.

The plate was then covered and left at room temperature in ambient light for several days until a thick mat of fresh non-sporulating mycelium had completely covered the top layer of liquid media.

The entire mycelial mat was lifted off the plate and transferred to a flat surface using sanitized forceps and patted dry with a paper towel lightly sprayed with 70% isopropyl alcohol. Approximately 1 g of wet mycelium was cut, transferred to a 1.5 ml microcentrifuge tube and macerated with the blunt end of a plastic inoculating loop.

Genomic DNA extraction

DNA extraction was then carried out from the prepared tissue using the Quick-DNA HMW MagBead Kit (Zymo, D6060, CA) with the following modifications to the protocol:

• 200 $μ$ l each of DNA Elution Buffer and Biofluid & Solid Tissue Buffer were used to accommodate the increased starting volume of solid tissue. There was no change to the proteinase K volume called for in the protocol.
• The sample was incubated for 24 hours in a water bath at 55 °C).
• The sample was macerated with a plastic micro-pestle to ensure good homogenization and tissue lysis after adding the initial buffers, midway through the initial incubation period, and finally prior to the initial centrifugation steps preceding DNA purification.
• The entire volume of supernatant was used to carry out the DNA purification steps and the volume of Quick-DNA MagBinding Buffer was increased to match the volume of supernatant used.
• DIY-friendly magbead seperator and vortex mixer were used to carry out the remaining steps of the DNA purification protocol.

DIY magnetic bead separator rack

A low-cost diy-friendly magnetic bead separator rack was constructed using packing tape, super strong neodymium disc magnets (32 mm diameter × 3 mm thick) and a cardboard box. A video demonstration is available on Figshare (Extended data³¹).

DIY vortex mixer

A vibrating back massager with a flat silicon head attachment was wrapped in a piece of rigid cardboard and affixed with tape to provide a contained area for the sample to vibrate. The massager was operated at low-speed for equivalent time periods in all steps requiring vortex mixing. While a lab-grade vortex mixer can be acquired at or below the cost of the massager used in this research, this device was already available to the researchers and further demonstrates the potential viability of alternative tools to those working in DIY lab environments. A video demonstration is available here (Extended data³²).

Nanopore sequencing and genome assembly

The initial size and concentration of the extracted genomic DNA was estimated using gel electrophoresis with 1% TBE gel at a fixed 45 V and a 1 kb ladder (Bioland Scientific, DM01-01, CA).

The microcentrifuge tube containing the extracted DNA was tightly wrapped in parafilm, put in a padded envelop and shipped via USPS Priority mail from Los Angeles, California to Toronto, Canada where it arrived 11 days later.

Approximately 1 $μ$ g was brought forward for library preparation. A DNA sequencing library was prepared with the SQK-LSK109 kit (Oxford Nanopore Technologies) according to the manufacturer’s protocol version GDE_9063_v109_revK_14Aug2019. Approximately 5 gigabases of sequencing data was collected using an R9.4.1 flow cell. Basecalling was performed with Guppy v5.015 in superior accuracy mode (Oxford Nanopore Technologies).

Sequencing quality was assessed using NanoPlot.¹³ All reads were used for assembly with Flye v2.9¹⁴ using the parameters –nano-hq, –genome-size=42m, –threads=16. A 5 kb contig was removed because it was determined it was not part of the genome after performing a web-BLAST search. Polishing was performed with 1 round of Racon v1.4.22¹⁵ and one round of medaka v1.4.4 (Oxford Nanopore Technologies), both with default settings. The assembly was annotated using Liftoff v1.6.1¹⁶ comparing against the Pestalotiopsis sp. NC0098 genome.¹⁷ Quality of the genome assembly was evaluated using BUSCO v5.2.2 using the sordiomycetes_odb10 database.¹⁸

To estimate the number of chromosomes, we used a previously described network-based approach.¹⁹ We identified the telomere repeat sequence as AACCCT from the initial assembly. All reads containing a minimum of three consecutive repeats of this sequence (or the reverse complement) were extracted using grep. Telomere-containing reads were aligned against each other in all-vs-all mode using minimap2.²⁰ Alignments were then filtered to retain only alignments with more than 95% query coverage using awk. A network graph was generated using the iGraph R package.²¹ The workflow is available on Zenodo (Analysis code).

Phylogenetic methods

A list of type and ex-type sequences containing ITS, TUB and TEF accessions for Pestalotiopsis, including an out-group using the species Neopestalotiopsis saprophytica, was constructed from prior work.²² The resulting phylogenetic analysis is shown in Figure 4.

Figure 4. Phylogenetic tree of the subject genome and related species based on maximum likelihood analysis of the combined ITS, TUB and TEF sequences with Neopestalotiopsis saprophytica used as the out-group.

The collection of 42 accessions were aligned with MAFFT v7.453²³ using the ‘–auto’ configuration command-line argument. The resulting alignments were trimmed and concatenated using Mega 11.²⁴

A Maximum Likelihood (ML) tree was constructed in Mega 11 using the combined ITS+TUB+TEF alignment with default settings and 100 bootstrap replications.

The final tree was saved in Newick format and visualized by rooting the tree on the Neopestalotiopsis saprophytica outgroup.

The phylogenetic analysis workflow, scripts and data is available on GitHub (Extended data).

Results

The Pestalotiopsis genome contains seven nuclear chromosomes

The majority of the DNA obtained from extraction was above 1 kb in length (Figure 3). After sequencing, we obtained a read N50 of 6.6 kb (Table 1), with approximately 105X coverage. Genome completion estimates revealed high completion, low duplication, and low fragmentation of the expected single-copy core genes. The total assembly length was 47.7 megabases, composed of five contigs with telomere repeats present at both the start and end of the contig, four contigs with no telomere repeats present, and a 68-kb circularized contig determined to be the full-length mitochondrial genome (Table 1). The BUSCO scores revealed high completion, with low missing and fragmented expected single-copy core genes.

Table 1. Genome assembly statistics by Flye and sequencing statistics reported by NanoPlot.

BUSCO scores (v5.2.2) were calculated using the sordaiomycetes_odb10 lineage, revealing 97.9%C (S:97.5%, D:0.4%, F:0.6%, M:1.5%, n:3817).

Sequencing and assembly statistics	Pestalotiopsis sp.
Total contigs	10
Largest contig	$8,884,865$
Total length	$47,674,442$
N₅₀	$6,008,314$
GC (%)	$51.79 %$
Read length N50	6578
Median read length	3936
Total bases	4,949,608,939
Median read quality	13
BUSCO score	97.5%C

After all sub-graphs were extracted from the network graph of telomere-containing reads, a total of 14 clusters containing telomere-containing reads were obtained (example of one is shown in Figure 5). Since each cluster represents one telomere and there are two telomeres per chromosome, this results in a total of seven linear nuclear chromosomes.

Figure 5. One of 14 telomere-containing read network graphs generated. Each purple dot is a single telomere-containing read, and each grey line connecting to another dot indicates a full-length alignment to another read.

This network graph shows the network of long reads that are derived from a single telomere.

The cost for consumables for this project was determined to be approximately $300 on a per-sample basis (Table 2), however, the total upfront cost would be approximately $3098.

Table 2. Cost of reagents and equipment in total and per sample.

Flow cell cost assumes 1/6 (i.e., 5 Gb of 30 Gigabases) of the throughput capacity was used.

Item	Total cost	Per sample
DNA extraction	$259	$3
LSK-SQK109	$599	$99
R9.4.1 Flow Cell	$900	$150
NEB Enzymes	$920	$38
Magnetic beads	$300	$7
Pipette tips, reagents	$120	$5
Total	$3098	$302

Phylogenetic analysis

A BLASTn search on the ITS, TUB and TEF regions of the isolate were obtained.

The ITS region showed similarities to P. grevilleae (NR_147548) with 99.5% (600/603), P. kenyana (NR_147549) with 99.83% (596/597), P. oryzae (NR_147547) with 99.83% (593/594), P. pini (MT374681) with 997% (601/606), and P. knightiae (KM199310) with 99.01% (599/605).

The TUB region showed similarities to P. photinicola (KY047662) with 98.83% (506/512), P. australasiae (KM199499) with 99.8% (491/492), P. trachicarpicola (JQ845946) with 98.05% (503/513), P. telopeae (KM199500) with 100% (481/481), and P. rosea (JX399069) with 96.88% (496/512).

The TEF region showed similarities to P. kenyana (KM199395) with 99.87% (774/775), P. oryzae (KM199398) with 99.87% (752/753), P. grevilleae (KM199407) with 97.25% (777/799), P. biciliata (KM199399) with 97.94% (762/778), P. knightiae (KM199408) 97.32% (763/784).

Discussion

Here, we obtained a high-quality genome assembly for Pestalatiopsis sp., comprising five contigs with telomeres at both ends and four contigs without telomere repeats. We also showed that obtaining fungal DNA suitable for Nanopore sequencing in a DIY lab environment is possible by modifying the protocol of a low-cost commercially available extraction kit. Genome sequencing is now becoming more accessible because of the relatively low capital requirements of the Oxford Nanopore MinION platform. In addition, the basecalling and consensus sequence quality are rapidly improving, resulting in high-quality, Nanopore-only genome assemblies for a variety of organisms.²⁵^,²⁶

While genome assemblers only report on the number and size of contiguous DNA sequences assembled, it is left up to the user to infer further information such as number of chromosomes. We used a previously developed approach¹⁹ based on network graphs to determine that 14 unique telomeres were present in the sequencing data. The DNA was extracted during a point in the life cycle where the genome is expected to be haploid. We therefore reason that seven unique telomere-capped chromosomes exist in this species, in addition to the circular mitochondrial genome.

One known quality concern with nanopore-only genome assemblies is the presence of frame-shifting indels that can affect protein prediction.²⁷ In this assembly, the high BUSCO completion score of 97.9% and low fragmentation score of the single-copy core genes (F:0.6%) over 3817 single-copy core genes suggest minimal frame-shifting indels were present after polishing. Until recently, it was often necessary to polish nanopore genome assemblies with high-quality Illumina reads using a tool like Pilon to obtain high-quality nanopore assemblies.²⁸ However, recent improvements to basecalling models and polishing algorithms enable high-quality genome assemblies from nanopore reads only. This further improves accessibility to high-quality genome assemblies by removing the need to sequence using an expensive platform, typically only found in labs with a large budget or institutional support.

With the exception of the nanopore library prep and sequencing, all organism isolation and culture work, genomic DNA extraction and microscopy was performed in a residential DIY home lab using consumer-grade or second-hand equipment acquired from consumer-facing online vendors like Amazon.com and eBay.com. This includes pipettors, a water bath, a thermocycler, a microcentrifuge, and a self-built laminar flow hood constructed with plywood, a 24x24x12 HEPA filter and inline duct fan. The cost of sequencing reagents for this genome was around $300; however, many of the reagents require the purchase of six to 24 reactions. All the software used in this project is freely available, thanks to the academic and open-source bioinformatics community.¹⁴^,¹⁵^,²⁰^,²⁹ Furthermore, all computation (except for basecalling) was performed on a consumer-grade laptop (Lenovo Thinkpad P14s) with 36 GB ram, AMD Ryzen 7 PRO 4750U processor (eight cores, 16 threads) and 1 TB NVMe SSD storage.

Pestalatiopsis sp. is known to possess diverse metabolic capabilities, including the production of potentially valuable secondary metabolites such as antitumor taxols,⁸ polyurethane-degrading hydrolases⁹ and compounds like alkaloids, polyketides, terpenoids, flavonoids, coumarins, xanthones, quinones, semiquinones, peptides, phenols, phenolic acids, and lactones.³⁰ This additional genome provides a high quality genome for another species, and a workflow to generate low cost genomes to the community for future studies in the metabolic capabilities of this genus.

Conclusions

Here, we generated a high-quality genome assembly using nanopore sequencing for Pestalatiopsis sp., and conclude that this species possesses seven nuclear chromosomes in addition to a mitochondrial genome. We conclude that genome sequencing for novel fungi species can be performed in a DIY environment for approximately USD$300 on a per-sample basis.

Data availability

Underlying data

The strain of Pestalotiopsis used in this research is available upon request from Josh McGinnis (josh@everymanbio.com).

BioProject: High-quality genome assembly of a Pestalotiopsis fungi, https://identifiers.org/bioproject: PRJNA773800

Extended data

Figshare: DIY Magnetic Bead Separator Rack, https://doi.org/10.6084/m9.figshare.19129856.v2.³¹

Figshare: Vibrating Back Massager as DIY Vortex Mixer, https://doi.org/10.6084/m9.figshare.19129862.v1.³²

Data are available under the terms of the Commons Attribution 4.0 International license (CC-BY 4.0).

Workflows, supporting scripts and accessions used in the phylogenetic analysis are available on GitHub (see below).

Analysis code

Analysis code available from: https://github.com/EverymanBio/pestalotiopsis

Archived analysis code as at time of publication: https://doi.org/10.5281/zenodo.6028895

License: GNU General Public License, version 3 (GPL-3.0).

Acknowledgements

We would like to acknowledge Damon Tighe for the makeshift-pestle idea, general DNA barcoding troubleshooting and reviewing. We’d also like to thank Kris Tatiossian, Ben Joris and Jos Houbraken for their feedback on this manuscript. We would like to thank Gregory B. Gloor for mentorship and providing funding for publication costs associated with this manuscript.

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Version 1

VERSION 1 PUBLISHED 20 Apr 2022

Author details Author details

¹ EverymanBio, Los Angeles, CA, 90047, USA
² Department of Biochemistry, University of Western Ontario, London, ON, Canada
³ Flow Genomics, Toronto, ON, Canada

Joshua L. McGinnis
Roles: Conceptualization, Data Curation, Formal Analysis, Funding Acquisition, Investigation, Methodology, Project Administration, Resources, Software, Supervision, Validation, Visualization, Writing – Original Draft Preparation, Writing – Review & Editing

Daniel J. Giguere
Roles: Conceptualization, Data Curation, Formal Analysis, Funding Acquisition, Investigation, Methodology, Project Administration, Resources, Software, Supervision, Validation, Visualization, Writing – Original Draft Preparation, Writing – Review & Editing

Competing interests

Joshua McGinnis founded EverymanBio, Daniel Giguere founded Flow Genomics

Grant information

DJG: Mitacs IT24796, Flow Genomics
The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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Published: 20 Apr 2022, 11:442

https://doi.org/10.12688/f1000research.110351.1

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© 2022 McGinnis JL and Giguere DJ. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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McGinnis JL and Giguere DJ. High-quality genome assembly of a Pestalotiopsis fungus using DIY-friendly methods [version 1; peer review: 3 approved with reservations]. F1000Research 2022, 11:442 (https://doi.org/10.12688/f1000research.110351.1)

NOTE: If applicable, it is important to ensure the information in square brackets after the title is included in all citations of this article.

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ApprovedThe paper is scientifically sound in its current form and only minor, if any, improvements are suggested

Approved with reservations A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.

Not approvedFundamental flaws in the paper seriously undermine the findings and conclusions

Version 1

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PUBLISHED 20 Apr 2022

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Reviewer Report 12 May 2022

Sinem Beyhan, Department of Infectious Diseases, J. Craig Venter Institute, La Jolla, CA, USA; Department of Medicine, University of California San Diego, La Jolla, CA, USA

Approved with Reservations

https://doi.org/10.5256/f1000research.121946.r135251

This is an exciting study of Pestalotiopsis sp. MUOB 440515 genome sequencing and assembly utilizing at home DIY research tools to isolate fungal genomic DNA and Nanopore long-read sequencing in a research lab. This study provides a good resource for ... Continue reading

This is an exciting study of Pestalotiopsis sp. MUOB 440515 genome sequencing and assembly utilizing at home DIY research tools to isolate fungal genomic DNA and Nanopore long-read sequencing in a research lab. This study provides a good resource for the other fungal researchers, as there are only 6 other genomes of Pestalotiopsis species deposited in NCBI. In addition, it is impressive that the researchers were able to obtain a high molecular weight DNA using make-shift tools, and were able to obtain long reads to generate telomere-to-telomere assemblies for most of the chromosomes (5 out of 7). Overall, I think this study would inspire other researchers to try and produce high quality genome assemblies. However, there are a couple of points that authors can improve on to make the manuscript more accessible to the reader.

In methods, strain description: Could you please describe how this strain was exactly isolated? Also, what is meant by “a wild-type clone of Laetiporus sp.”? Were these two strains growing together on an aged hardwood stump? In addition, I was able to get the isolate name (MUOB 440515), but it is not obvious throughout the text. Could you please use the isolate name as appropriate?
Strain sharing: The strain needs to be deposited to a publicly accessible strain collection (i.e. ATCC or BEI)
Figure 1: Please include scale bars in all panels.
Figure 4 and related analysis: The “subject” needs to be relabeled as Pestalotiopsis sp. MUOB 440515. All the strains used for this phylogenetic analysis need to be described better in the methods. The previously published work can be cited but a metadata of all the strains needs to be included.
Figure 5 and related analysis: I am wondering if there is a better way of presenting this data. Can you align these telomere containing reads onto the large contigs and see if you can extend the chromosome hands manually?
Data sharing: Could the authors include genome annotation in the bioproject?
Overall: As stated multiple times throughout the manuscript, one of the premises here is to provide a low cost sequencing method for other fungal researchers. However, I am not convinced that the manuscript is providing an improvement in lowering the cost. If one were to calculate per sample cost, as the authors did here, I would argue that some researchers would calculate a lower cost depending on the institution. So, the statements about the cost/sample should be revised.
Overall: The methodology as it is described in the abstract and the introduction is misleading. The authors overemphasize the use of DIY tools and at home protocols, but I think the sequencing library preparation and the ONT sequencing were done in a research facility. I am impressed by the quality of the HMW gDNA isolated using a home lab, but I think the statements about a home lab being suitable for this study should be revised and confined to the HMW gDNA isolation portion.

Is the work clearly and accurately presented and does it cite the current literature?

Partly
Is the study design appropriate and is the work technically sound?

Yes
Are sufficient details of methods and analysis provided to allow replication by others?

Partly
If applicable, is the statistical analysis and its interpretation appropriate?

Not applicable
Are all the source data underlying the results available to ensure full reproducibility?

Yes
Are the conclusions drawn adequately supported by the results?

Yes

Competing Interests: No competing interests were disclosed.

Reviewer Expertise: Fungal genomics and pathogenesis

I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.

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25

Reviewer Report 10 May 2022

Huzefa A Raja, Department of Chemistry and Biochemistry, University of North Carolina at Greensboro, Greensboro, NC, USA

Approved with Reservations

https://doi.org/10.5256/f1000research.121946.r136304

It seems like a fun project to do. I appreciate the attempts made to isolate a fungus and sequence its genome. I am not sure I am qualified to review the genomics/bioinformatics section of the manuscript. I am not an ... Continue reading

It seems like a fun project to do. I appreciate the attempts made to isolate a fungus and sequence its genome. I am not sure I am qualified to review the genomics/bioinformatics section of the manuscript. I am not an expert in bioinformatics analysis of fungal genomes. I will focus only on the mycology aspects of this paper, with some comments on the molecular biology section on HMW DNA extraction. I want to provide these comments so the manuscript can be indexed, but it needs some work.

Comments below are on the mycology aspects:

How was the fungus isolated? Please give more details.
"A double-layer media plate was prepared using a 9 mm Petri dish with a 30 ml Malt Extract Agar (MEA) bottom-layer and a 20 ml Potato Dextrose broth top-layer" – Please can you elaborate more on what was the purpose? Perhaps a figure of this in the Appendix or Supplementary section.
Why did the authors PCR gene regions again? They could have tried to extract the regions from the whole genome. I can see sequencing the ITS region since it is multi-copy and could be hard to extract from the whole genome. Just wondering. I have no problem with that.
In the methods, the use of the word “clone” is incorrect in the context used.
Figure 1B can you provide a scale bar.
Provide a citation for the PCR amplification program used.
Overall, I see the manuscript has focused a lot on at-home isolation of DNA and genome sequencing and that is fine. I am not sure the figure of an Agarose gel electrophoresis is necessary in the main manuscript. The modified protocols of the DNA extraction kit might be well suited in an Appendix or Supplementary section.
Phylogenetic analysis needs major work:
- a. I cannot tell which isolates of Pestalotiopsis were used from GenBank. All the metadata is missing. The authors should present a table with all of those data and provide the isolate number of the species besides the binomial.
- b. Please provide a Maximum Likelihood tree with branch lengths and Bootstrap branch support values ≥ 75 % are shown above the nodes. Bootstrap replicates should be at least 1000, not 100.
- c. What was the rationale for using ITS, TUB, and TEF genes?
- d. Did the authors do a BLAST search against type data?
- e. Did the authors submit the Sanger sequencing data to GenBank? Tree alignment and tree into TreeBase? Was the culture submitted to a culture collection? In the legend for Figure 4, the authors need to give how many bp were used, how many strains, bootstrap values above the nodes, etc.
"sp." in species should not be in italics throughout the manuscript, please check.
Maybe better to give all equipment used and their vendors in a separate table format in the Appendix of the methods? Make sure all specific details are provided.
In Table 2, the total incurred cost and cost per sample are provided. Can you provide a comparison between the cost incurred for this project and how much an academic lab spends for the same?
I am left wondering after the 3-gene phylogeny and ITS Barcoding work, and genome sequencing of the fungus: did the authors ascertain the species identity of the fungus? Or do they think it is new to science and if so why? Please mention in the manuscript. Also, you have to remember that Pestalotiopsis is a very large genus, with many species with no sequence data. Based on this work I would not be able to call it new unless a thorough species comparison is performed.
"Sordariomycetes" is spelled incorrectly in Table 1.

Questions/Comments about the genome sequencing:

Did the authors submit the whole genome data to GenBank?
For the HMW genomic DNA, what was the Nanodrop or Qubit reading? How much total DNA was obtained and how much per µl? I am curious about how much HMW DNA they obtained and what was the weight of the DNA.

I have seen the enthusiasm of the first author in working with fungi, and I applaud their attempts to write their workup. Above I try to provide some comments to strengthen the mycology section of the manuscript. It is a good manuscript and I recommend revisions to improve the quality of the work and re-organization of sections to aid in clarity. Remove all detailed methods into the Appendix or Supplementary section.

Is the work clearly and accurately presented and does it cite the current literature?

Yes
Is the study design appropriate and is the work technically sound?

Yes
Are sufficient details of methods and analysis provided to allow replication by others?

Yes
If applicable, is the statistical analysis and its interpretation appropriate?

Partly
Are all the source data underlying the results available to ensure full reproducibility?

Partly
Are the conclusions drawn adequately supported by the results?

Yes

Competing Interests: No competing interests were disclosed.

Reviewer Expertise: My research focuses on freshwater ascomycete fungal diversity, distribution and phylogeny. I work on isolating and describing these fungi, thus contributing to the origins and diversity of this important ecological group of fungi. My research findings have important implications in studies of biodiversity, phylogenetics and taxonomic studies of the kingdom fungi. I also work with natural products chemists on the chemical mycology of freshwater fungi and fungal endophytes.

I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.

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35

Reviewer Report 09 May 2022

Paula Maria Moolhuijzen, Center for Crop Disease and Management, School of Molecular and Life Sciences, Curtin University, Bentley, Australia

Approved with Reservations

https://doi.org/10.5256/f1000research.121946.r135248

This brief report describes the culture, isolation, DNA extraction, genome sequencing and assembly of an unknown Pestalotiopsis species (fungal isolate MUOB 440515). The study utilised Oxford Nanopore Technology (ONT) long read sequence at +100x coverage to obtain telomere-to-telomere contig assembly ... Continue reading

This brief report describes the culture, isolation, DNA extraction, genome sequencing and assembly of an unknown Pestalotiopsis species (fungal isolate MUOB 440515). The study utilised Oxford Nanopore Technology (ONT) long read sequence at +100x coverage to obtain telomere-to-telomere contig assembly for 5 of the 7 chromosomes that were estimated also in this study.

Reviewer main comment:
This is quite an impressive fungal genome assembly (10 contigs) and a useful resource, especially as only 7 others genomes have been sequenced for this genus. The main focus of the study however appears to be the costing of samples based on a do-it-yourself (DIY) home laboratory, spruiking affordable sample sequencing. I think the authors could improve this report by highlighting more the scientific benefits of affordable sequencing and the quality of this assembly in comparison to what is currently available.

Other comments and recommendations:

Please make it clearer in the methods that the Nanopore library preparation, sequencing and base calling was all done elsewhere. This is not clear until the discussion.
The authors provide video demonstrations, analysis code and have published the genome in NCBI GenBank. Unfortunately, the published genome is not annotated, can the authors clarify in the manuscript where to find the annotations.
Please use the isolate identifier “Pestalotiopsis sp. isolate MUOB 440515”. Do not refer to the isolate as the subject.

Section: Abstract:

There are over 3,500 genomes in NCBI GenBank. Can this be updated?
“The project (costing $300)”. The low cost of ONT platform sequencing is already mentioned. This number can be misleading, the project costs at least $3000. The cost per sample is potentially $300 if the user multiplexes samples, which is not done here. Can the authors please remove “(costing $300)” from the abstract?

Section: Introduction:

“recently developed MinION nanopore”. I think the authors can say "The MinION has been commercially available since 2015".
“Nanopore Technologies provides both the hardware and reagents needed for genome sequencing for $1000 USD”. This is the cost of the starter kit. Can the authors confirm this here?

Section: Methods:

“prior to the initial centrifugation steps”. Was a centrifuge used and what does this equipment cost?

Section: Nanopore sequencing and genome assembly:

“...shipped via USPS Priority mail from Los Angeles, California to Toronto, Canada where it arrived 11 days later”. Who in Toronto, Canada did the ONT sequencing? Please add to methods.
“Basecalling was performed with Guppy v5.015 in superior accuracy mode (Oxford Nanopore Technologies).” What software was used during the sequencing? Was ONT minKNOW used? What were the computer specifications used for the collection of data, base calling and analysis?
Figure 3 and Figure 4 belong in the results section.

Section: Results:
Section: The Pestalotiopsis genome contains seven nuclear chromosomes:

Referring to the title of this section, is this the first time the number of chromosomes has been estimated for this genus? If it is a first finding please highlight this.
Did the authors assign the full length contigs to chromosomes for this isolate? If so, please state the which chromosomes are full length. Can the authors check the genome synteny to the other sequenced species?
How do the Table 1 assembly metrics compare to other related genomes in the genus? Can the authors please comment on this?
First two paragraphs are a bit jumbled. Can the authors describe the genome assembly first and then describe the genes?
“The cost for consumables for this project was determined to be approximately $300 on a per-sample basis (Table 2), however, the total upfront cost would be approximately $3098.”
This is only a potential cost as multiplexed samples were not run. The MinION yield of 30 Gb is only an estimate. The yield obtained in this study was 5 Gb, 1/6^th of the estimated total yield, but Table 2 has one sample as 1/10^th of the consumables cost? Can the authors clarify? Can the authors add a statement that this does not include the actual MinION device?
Can the authors please add a column to Table 2 for item description?
Typo in Table 2. Change “LSK-SQK109” to “SQK-LSK109”.

Section: Phylogenetic analysis:

No results are presented for the phylogenetic tree created, only blast results. Can the authors please include the tree results in this section.
Can the authors describe the blast results better? Example: "The isolate MUOB 440515 ITS region had high nucleotide sequence identity to …"
Typo, change 997% to 99.7%.

Section: Discussion:

”However, recent improvements to basecalling models and polishing algorithms enable high-quality genome assemblies from nanopore reads only.” Please add references.
“This further improves accessibility to high-quality genome assemblies by removing the need to sequence using an expensive platform, typically only found in labs with a large budget or institutional support.” But didn’t the authors rely on institutional support for the library preparation, sequencing and base calling? Can the authors please reword this sentence to make it clearer that Illumina sequencing for polishing ONT genomes is not required at xx depth of coverage and include references?
”With the exception of the nanopore library prep and sequencing”. This should be made clearer in the methods section. I am a little surprised the nanopore sequencing wasn’t done in house, as I think it would be doable?
“Furthermore, all computation (except for basecalling) was performed on a consumer-grade laptop (Lenovo Thinkpad P14s) with 36 GB ram, AMD Ryzen 7 PRO 4750U processor (eight cores, 16 threads) and 1 TB NVMe SSD storage.” This should be in the methods, but the authors can still discuss base calling and assembly computational requirements.
Last paragraph of this section. Can the authors discuss this isolate's position in the phylogenetic tree or how it compares to the closest sequenced species?

Section: Conclusion:
The conclusion reads like a flyer for a business. In the conclusion can the authors highlight the academic merits provided by this study? For example, maybe describe the scientific benefits that can be derived from affordable genome sequencing?

Is the work clearly and accurately presented and does it cite the current literature?

Partly
Is the study design appropriate and is the work technically sound?

Yes
Are sufficient details of methods and analysis provided to allow replication by others?

Yes
If applicable, is the statistical analysis and its interpretation appropriate?

Not applicable
Are all the source data underlying the results available to ensure full reproducibility?

Yes
Are the conclusions drawn adequately supported by the results?

Partly

Competing Interests: No competing interests were disclosed.

Reviewer Expertise: Genomics

I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.

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Paula Maria Moolhuijzen, Curtin University, Bentley, Australia
Huzefa A Raja, University of North Carolina at Greensboro, Greensboro, USA
Sinem Beyhan, J. Craig Venter Institute, La Jolla, USA; University of California San Diego, La Jolla, USA

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Reviewer Report

21 Views

12 May 2022 | for Version 1

Sinem Beyhan, Department of Infectious Diseases, J. Craig Venter Institute, La Jolla, CA, USA; Department of Medicine, University of California San Diego, La Jolla, CA, USA

21 Views Cite this report Responses(0)

Approved With Reservations

This is an exciting study of Pestalotiopsis sp. MUOB 440515 genome sequencing and assembly utilizing at home DIY research tools to isolate fungal genomic DNA and Nanopore long-read sequencing in a research lab. This study provides a good resource for the other fungal researchers, as there are only 6 other genomes of Pestalotiopsis species deposited in NCBI. In addition, it is impressive that the researchers were able to obtain a high molecular weight DNA using make-shift tools, and were able to obtain long reads to generate telomere-to-telomere assemblies for most of the chromosomes (5 out of 7). Overall, I think this study would inspire other researchers to try and produce high quality genome assemblies. However, there are a couple of points that authors can improve on to make the manuscript more accessible to the reader.

In methods, strain description: Could you please describe how this strain was exactly isolated? Also, what is meant by “a wild-type clone of Laetiporus sp.”? Were these two strains growing together on an aged hardwood stump? In addition, I was able to get the isolate name (MUOB 440515), but it is not obvious throughout the text. Could you please use the isolate name as appropriate?
Strain sharing: The strain needs to be deposited to a publicly accessible strain collection (i.e. ATCC or BEI)
Figure 1: Please include scale bars in all panels.
Figure 4 and related analysis: The “subject” needs to be relabeled as Pestalotiopsis sp. MUOB 440515. All the strains used for this phylogenetic analysis need to be described better in the methods. The previously published work can be cited but a metadata of all the strains needs to be included.
Figure 5 and related analysis: I am wondering if there is a better way of presenting this data. Can you align these telomere containing reads onto the large contigs and see if you can extend the chromosome hands manually?
Data sharing: Could the authors include genome annotation in the bioproject?
Overall: As stated multiple times throughout the manuscript, one of the premises here is to provide a low cost sequencing method for other fungal researchers. However, I am not convinced that the manuscript is providing an improvement in lowering the cost. If one were to calculate per sample cost, as the authors did here, I would argue that some researchers would calculate a lower cost depending on the institution. So, the statements about the cost/sample should be revised.
Overall: The methodology as it is described in the abstract and the introduction is misleading. The authors overemphasize the use of DIY tools and at home protocols, but I think the sequencing library preparation and the ONT sequencing were done in a research facility. I am impressed by the quality of the HMW gDNA isolated using a home lab, but I think the statements about a home lab being suitable for this study should be revised and confined to the HMW gDNA isolation portion.

Is the work clearly and accurately presented and does it cite the current literature?

Partly
Is the study design appropriate and is the work technically sound?

Yes
Are sufficient details of methods and analysis provided to allow replication by others?

Partly
If applicable, is the statistical analysis and its interpretation appropriate?

Not applicable
Are all the source data underlying the results available to ensure full reproducibility?

Yes
Are the conclusions drawn adequately supported by the results?

Yes

Competing Interests

No competing interests were disclosed.

Reviewer Expertise

Fungal genomics and pathogenesis

I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.

Respond to this report

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Reviewer Report

25 Views

10 May 2022 | for Version 1

Huzefa A Raja, Department of Chemistry and Biochemistry, University of North Carolina at Greensboro, Greensboro, NC, USA

25 Views Cite this report Responses(0)

Approved With Reservations

It seems like a fun project to do. I appreciate the attempts made to isolate a fungus and sequence its genome. I am not sure I am qualified to review the genomics/bioinformatics section of the manuscript. I am not an expert in bioinformatics analysis of fungal genomes. I will focus only on the mycology aspects of this paper, with some comments on the molecular biology section on HMW DNA extraction. I want to provide these comments so the manuscript can be indexed, but it needs some work.

Comments below are on the mycology aspects:

How was the fungus isolated? Please give more details.
"A double-layer media plate was prepared using a 9 mm Petri dish with a 30 ml Malt Extract Agar (MEA) bottom-layer and a 20 ml Potato Dextrose broth top-layer" – Please can you elaborate more on what was the purpose? Perhaps a figure of this in the Appendix or Supplementary section.
Why did the authors PCR gene regions again? They could have tried to extract the regions from the whole genome. I can see sequencing the ITS region since it is multi-copy and could be hard to extract from the whole genome. Just wondering. I have no problem with that.
In the methods, the use of the word “clone” is incorrect in the context used.
Figure 1B can you provide a scale bar.
Provide a citation for the PCR amplification program used.
Overall, I see the manuscript has focused a lot on at-home isolation of DNA and genome sequencing and that is fine. I am not sure the figure of an Agarose gel electrophoresis is necessary in the main manuscript. The modified protocols of the DNA extraction kit might be well suited in an Appendix or Supplementary section.
Phylogenetic analysis needs major work:
- a. I cannot tell which isolates of Pestalotiopsis were used from GenBank. All the metadata is missing. The authors should present a table with all of those data and provide the isolate number of the species besides the binomial.
- b. Please provide a Maximum Likelihood tree with branch lengths and Bootstrap branch support values ≥ 75 % are shown above the nodes. Bootstrap replicates should be at least 1000, not 100.
- c. What was the rationale for using ITS, TUB, and TEF genes?
- d. Did the authors do a BLAST search against type data?
- e. Did the authors submit the Sanger sequencing data to GenBank? Tree alignment and tree into TreeBase? Was the culture submitted to a culture collection? In the legend for Figure 4, the authors need to give how many bp were used, how many strains, bootstrap values above the nodes, etc.
"sp." in species should not be in italics throughout the manuscript, please check.
Maybe better to give all equipment used and their vendors in a separate table format in the Appendix of the methods? Make sure all specific details are provided.
In Table 2, the total incurred cost and cost per sample are provided. Can you provide a comparison between the cost incurred for this project and how much an academic lab spends for the same?
I am left wondering after the 3-gene phylogeny and ITS Barcoding work, and genome sequencing of the fungus: did the authors ascertain the species identity of the fungus? Or do they think it is new to science and if so why? Please mention in the manuscript. Also, you have to remember that Pestalotiopsis is a very large genus, with many species with no sequence data. Based on this work I would not be able to call it new unless a thorough species comparison is performed.
"Sordariomycetes" is spelled incorrectly in Table 1.

Questions/Comments about the genome sequencing:

Did the authors submit the whole genome data to GenBank?
For the HMW genomic DNA, what was the Nanodrop or Qubit reading? How much total DNA was obtained and how much per µl? I am curious about how much HMW DNA they obtained and what was the weight of the DNA.

I have seen the enthusiasm of the first author in working with fungi, and I applaud their attempts to write their workup. Above I try to provide some comments to strengthen the mycology section of the manuscript. It is a good manuscript and I recommend revisions to improve the quality of the work and re-organization of sections to aid in clarity. Remove all detailed methods into the Appendix or Supplementary section.

Is the work clearly and accurately presented and does it cite the current literature?

Yes
Is the study design appropriate and is the work technically sound?

Yes
Are sufficient details of methods and analysis provided to allow replication by others?

Yes
If applicable, is the statistical analysis and its interpretation appropriate?

Partly
Are all the source data underlying the results available to ensure full reproducibility?

Partly
Are the conclusions drawn adequately supported by the results?

Yes

Competing Interests

No competing interests were disclosed.

Reviewer Expertise

My research focuses on freshwater ascomycete fungal diversity, distribution and phylogeny. I work on isolating and describing these fungi, thus contributing to the origins and diversity of this important ecological group of fungi. My research findings have important implications in studies of biodiversity, phylogenetics and taxonomic studies of the kingdom fungi. I also work with natural products chemists on the chemical mycology of freshwater fungi and fungal endophytes.

I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.

Respond to this report

Responses (0)

Back to all reports

Reviewer Report

35 Views

09 May 2022 | for Version 1

Paula Maria Moolhuijzen, Center for Crop Disease and Management, School of Molecular and Life Sciences, Curtin University, Bentley, Australia

35 Views Cite this report Responses(0)

Approved With Reservations

This brief report describes the culture, isolation, DNA extraction, genome sequencing and assembly of an unknown Pestalotiopsis species (fungal isolate MUOB 440515). The study utilised Oxford Nanopore Technology (ONT) long read sequence at +100x coverage to obtain telomere-to-telomere contig assembly for 5 of the 7 chromosomes that were estimated also in this study.

Reviewer main comment:
This is quite an impressive fungal genome assembly (10 contigs) and a useful resource, especially as only 7 others genomes have been sequenced for this genus. The main focus of the study however appears to be the costing of samples based on a do-it-yourself (DIY) home laboratory, spruiking affordable sample sequencing. I think the authors could improve this report by highlighting more the scientific benefits of affordable sequencing and the quality of this assembly in comparison to what is currently available.

Other comments and recommendations:

Please make it clearer in the methods that the Nanopore library preparation, sequencing and base calling was all done elsewhere. This is not clear until the discussion.
The authors provide video demonstrations, analysis code and have published the genome in NCBI GenBank. Unfortunately, the published genome is not annotated, can the authors clarify in the manuscript where to find the annotations.
Please use the isolate identifier “Pestalotiopsis sp. isolate MUOB 440515”. Do not refer to the isolate as the subject.

Section: Abstract:

There are over 3,500 genomes in NCBI GenBank. Can this be updated?
“The project (costing $300)”. The low cost of ONT platform sequencing is already mentioned. This number can be misleading, the project costs at least $3000. The cost per sample is potentially $300 if the user multiplexes samples, which is not done here. Can the authors please remove “(costing $300)” from the abstract?

Section: Introduction:

“recently developed MinION nanopore”. I think the authors can say "The MinION has been commercially available since 2015".
“Nanopore Technologies provides both the hardware and reagents needed for genome sequencing for $1000 USD”. This is the cost of the starter kit. Can the authors confirm this here?

Section: Methods:

“prior to the initial centrifugation steps”. Was a centrifuge used and what does this equipment cost?

Section: Nanopore sequencing and genome assembly:

“...shipped via USPS Priority mail from Los Angeles, California to Toronto, Canada where it arrived 11 days later”. Who in Toronto, Canada did the ONT sequencing? Please add to methods.
“Basecalling was performed with Guppy v5.015 in superior accuracy mode (Oxford Nanopore Technologies).” What software was used during the sequencing? Was ONT minKNOW used? What were the computer specifications used for the collection of data, base calling and analysis?
Figure 3 and Figure 4 belong in the results section.

Section: Results:
Section: The Pestalotiopsis genome contains seven nuclear chromosomes:

Referring to the title of this section, is this the first time the number of chromosomes has been estimated for this genus? If it is a first finding please highlight this.
Did the authors assign the full length contigs to chromosomes for this isolate? If so, please state the which chromosomes are full length. Can the authors check the genome synteny to the other sequenced species?
How do the Table 1 assembly metrics compare to other related genomes in the genus? Can the authors please comment on this?
First two paragraphs are a bit jumbled. Can the authors describe the genome assembly first and then describe the genes?
“The cost for consumables for this project was determined to be approximately $300 on a per-sample basis (Table 2), however, the total upfront cost would be approximately $3098.”
This is only a potential cost as multiplexed samples were not run. The MinION yield of 30 Gb is only an estimate. The yield obtained in this study was 5 Gb, 1/6^th of the estimated total yield, but Table 2 has one sample as 1/10^th of the consumables cost? Can the authors clarify? Can the authors add a statement that this does not include the actual MinION device?
Can the authors please add a column to Table 2 for item description?
Typo in Table 2. Change “LSK-SQK109” to “SQK-LSK109”.

Section: Phylogenetic analysis:

No results are presented for the phylogenetic tree created, only blast results. Can the authors please include the tree results in this section.
Can the authors describe the blast results better? Example: "The isolate MUOB 440515 ITS region had high nucleotide sequence identity to …"
Typo, change 997% to 99.7%.

Section: Discussion:

”However, recent improvements to basecalling models and polishing algorithms enable high-quality genome assemblies from nanopore reads only.” Please add references.
“This further improves accessibility to high-quality genome assemblies by removing the need to sequence using an expensive platform, typically only found in labs with a large budget or institutional support.” But didn’t the authors rely on institutional support for the library preparation, sequencing and base calling? Can the authors please reword this sentence to make it clearer that Illumina sequencing for polishing ONT genomes is not required at xx depth of coverage and include references?
”With the exception of the nanopore library prep and sequencing”. This should be made clearer in the methods section. I am a little surprised the nanopore sequencing wasn’t done in house, as I think it would be doable?
“Furthermore, all computation (except for basecalling) was performed on a consumer-grade laptop (Lenovo Thinkpad P14s) with 36 GB ram, AMD Ryzen 7 PRO 4750U processor (eight cores, 16 threads) and 1 TB NVMe SSD storage.” This should be in the methods, but the authors can still discuss base calling and assembly computational requirements.
Last paragraph of this section. Can the authors discuss this isolate's position in the phylogenetic tree or how it compares to the closest sequenced species?

Section: Conclusion:
The conclusion reads like a flyer for a business. In the conclusion can the authors highlight the academic merits provided by this study? For example, maybe describe the scientific benefits that can be derived from affordable genome sequencing?

Is the work clearly and accurately presented and does it cite the current literature?

Partly
Is the study design appropriate and is the work technically sound?

Yes
Are sufficient details of methods and analysis provided to allow replication by others?

Yes
If applicable, is the statistical analysis and its interpretation appropriate?

Not applicable
Are all the source data underlying the results available to ensure full reproducibility?

Yes
Are the conclusions drawn adequately supported by the results?

Partly

Competing Interests

No competing interests were disclosed.

Reviewer Expertise

Genomics

I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.

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High-quality genome assembly of a Pestalotiopsis fungus using DIY-friendly methods

Abstract

Keywords

Introduction

Methods

Strain and culture conditions

Figure 1. Microscopic images of Pestalotiopsis used in this study.

DNA extraction

Figure 2. Collection of do-it-yourself (DIY) equipment used in this project.

Figure 3. Estimated molecular weight of DNA.

PCR reaction

Sequencing

High molecular weight genomic extraction

Nanopore sequencing and genome assembly

Phylogenetic methods

Figure 4. Phylogenetic tree of the subject genome and related species based on maximum likelihood analysis of the combined ITS, TUB and TEF sequences with Neopestalotiopsis saprophytica used as the out-group.

Results

The Pestalotiopsis genome contains seven nuclear chromosomes

Table 1. Genome assembly statistics by Flye and sequencing statistics reported by NanoPlot.

Figure 5. One of 14 telomere-containing read network graphs generated. Each purple dot is a single telomere-containing read, and each grey line connecting to another dot indicates a full-length alignment to another read.

Table 2. Cost of reagents and equipment in total and per sample.

Phylogenetic analysis

Discussion

Conclusions

Data availability

Underlying data

Extended data

Analysis code

Acknowledgements

References

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