A draft reference assembly of the Psilocybe cubensis genome

This genomic archive paper (2021) contains the entire genetic sequence of the Psilocybe cubensis mushroom, more specifically the genome cluster for psilocybin synthesis.

Abstract

“We describe the use of high-fidelity single molecule sequencing to assemble the genome of the psychoactive Psilocybe cubensis mushroom. The genome is 46.6Mb, 46% GC, and in 32 contigs with an N50 of 3.3Mb. The BUSCO completeness scores are 97.6% with 1.2% duplicates. The Psilocybin synthesis cluster exists in a single 3.2Mb contig. The dataset is available from NCBI BioProject with accessions PRJNA687911 and PRJNA700437.”

Authors: Kevin McKernan, Liam T Kane, Seth Crawford, Chen-Shan Chin, Aaron Trippe, Stephen McLaughlin

Summary

Abstract

The genome of the psychoactive Psilocybe cubensis mushroom was assembled using high-fidelity single molecule sequencing and has a completeness score of 97.6%.

Kevin McKernan conceptualized the project, gathered data, developed the methodology, and wrote the paper.

Introduction

There are hundreds of mushrooms capable of synthesizing the psychoactive compound psilocybin, but no public references exist in NCBI with N50s longer than 50kb.

DNA isolation

Dried stems from Psilocybe cubensis strain P.envy were ground to a fine powder and incubated with RNase A, Proteinase K and cetrimonium bromide for 1 hour, 5 minutes at 14000 rpm and 600 uL chloroform for 5 minutes, until the samples became opaque. 400 uL of MIP (marijuana infused products) Solution B and 400 uL DNA Binding Beads were added to an Eppendorf tube and incubated at room temperature for 15 minutes. The supernatant was removed and the beads were washed twice with 70% ethanol and allowed to dry for 5 minutes.

Sequencing

Sequencing libraries were constructed and 773,735 CCS reads were generated. Quast was used to assess the quality of the input and output sequence files.

Assembly and annotation

The unfiltered CCS data was assembled using the Peregrine assembler and 13,478 genes were annotated using FunAnnotate v1.8.4. 96-98.75% of the sequences from three other public Psilocybe cubensis datasets align to the new HiFi generated P.envy genome.

Psilocybe cubensis P.envy reference genome was aligned using minimap2 and bwa-mem, and whole genome alignment plots were visualized using MUMmer and Integrative Genomics Viewer.

Psilocybe cubensis P.envy genome assembly and annotation, based on Illumina reads, hosted at JGI, and Medicinal Genomics P. cubensis strain PRJNA687911.

A total of 553,716 variants were identified using GATK HaplotypeCaller (version 4.1.6.0) and converted from gff3 format into SnpEff database. Of these, 375,896 (67.9%) are heterozygous and 177,820 (32.1%) are homozygous with a ratio of just over 2 to 1 heterozygous:homozygous variants.

Structural variation

The N-methyltransferase gene in P.envy contains a structural variation not seen in previous P. cubensis surveys. This variation extends the 3′ end of the gene and may be important for Psilocybin production.

Data availability

Variant calling scripts, annotated variants and the gff3 file used to perform the annotation analysis are available for download.

Current Peer Review Status:

The authors have addressed all comments and requested edits, and have improved the manuscript. One typo remains.

1 Egret Bioscience Ltd., West Kelowna, Canada 2 Lighthouse Genomics Inc., BC, Canada

McKernan and colleagues present on the first highly contiguous draft genome for the magic mushroom Psilocybe cubensis. The authors acknowledge that fungi can have multiple nuclei in a cell, sometimes with completely different haplotypes, and suggest that their assembly could be a metagenome assembly.

The Penis Envy strain of P. cubensis was supposedly selected from an amazonian accession, and was more potent than most other P. cubensis strains, hinting at a mutation in the psiK gene.

The mechanism behind the higher perceived potency of PE could be polymorphisms at other loci involved in the psilocybin biosynthetic pathways as well as ancillary genes involved in the SAM salvage pathway.

Node Position Target gene Ontogeny

NODE_599 19295 sahH Sadenosyllhomocysteine hydrolase NODE_599 19304 sahH Sadenosyllhomocysteine hydrolase NODE_599 19415 sahH Sadenosyllhomocysteine hydrolase NODE_599 19744 sahH Saden NODE_6392 314122 samS Sadenosylmethionine synthetase NODE_712 1234504 metS lmethionine synthetase NODE_755 63920 psiD tryptophan decarboxylase NODE_755 64575 psiD tryptophan decarboxylase NODE_

Summary:

The manuscript presents an assembly of the fungus Psilocybe cubensis, and notes a potentially important structural variation in the psilocybin N-methyltransferase gene.

A high quality reference genome for the species was undertaken using Pacific Biosciences Sequel II HiFi methods. Single nucleotide polymorphisms were called with appropriate software, but parameters were not detailed in the text.

Figure 1 and both tables are clear and informative, but Figure 3 could be improved by marking the “tracks” clearly, but numbering and perhaps with additional labels for RNAseq, P. cyanescens, and annotation.

Other comments:

The title might be better as “A draft reference assembly of…”, the introduction might be better stated as “about 200 mushroom species”, and the citations are incomplete in the last sentence of “Assembly and annotation” section.