Stephen Douglas Russell

This protocol is for dA-tailing, which is an enzymatic method for adding a non-templated nucleotide to the 3' end of a blunt, double-stranded DNA molecule. In other words, this puts a single adenine nucleotide at each 3′ end of our PCR product, creating a site for the ligation adapter to attach to. The module also repairs ends and provides 5′ phosphorylation. Simple process - create a reaction with three chemicals, cleanup the product with beads. Time required: ~45 minutes The NEB protocol this is based on can be found here.

Overview: The goals of this protocol are to pool subsamples of all your PCR products into a single pooled library tube and to purify that product using magnetic beads. Philosophical note: Equal-volume pooling does not normalize DNA contributions between samples. We do not make an attempt to normalize each PCR reaction concentrations as a part of this protocol. Time required: ~45 minutes (mostly waiting)

This process will take your dA-tailed library and add the nanopore adapters. Simply combine several chemicals for a single reaction and do a bead cleanup. Tested with: Flowcells: MinION 10.4.1 Ligation Kit: V14 - LSK114 Time required: ~45 minutes

Overview: This protocol describes the steps used to load a 10.4.1 MinION flowcell utilizing the Q20+ (V14) Ligation Sequencing Kit from ONT. This protocol has been tested with MinION R10.4.1 flowcells. Time required: 10-15 minutes with a five and 40 minute wait in the middle.

To better understand the taxonomic identity and diversity of commercially available mushrooms in North America, specimens were solicited from commercial businesses such as grocery stores, markets, apothecaries, and online retailers. Two hundred and thirty-eight specimens were submitted by sixteen contributors. The majority of specimens were dried food products (179, ~66%) but canned/pickled (29) or fresh food (30) products and dried medicinal products (22) were also submitted. About 32% of the specimens were successfully barcoded, consisting of either dried or fresh food products. Almost no medicinal products or canned/pickled items yielded successful results, but modifications that may increase success rates are presented. Gross misidentifications and discrepancies between the information on regulatory labeling and food contents were found based on ITS barcode identification. Brief recommendations for ingredient labeling are provided.

Two novel boletes, Gyroporus binkatensis and Cyanoboletus sulcatipes , are described from District Swat, Pakistan, based on detailed macro‐ and micromorphological observations and phylogenetic analysis derived from Oxford Nanopore sequencing. G. binkatensis is characterized by an orbicular, dull reddish‐brown pileus with upturned and rimose margin, an eccentric tapering stipe, a hymenophore that remains white upon bruising, and oblong to subcylindrical basidiospores. C. sulcatipes exhibits a dull, dry, smooth pileus, irregular elongated pores that become irpicoid to daedaloid with age, a stipe distinctly longitudinally sulcate at the apex, narrowly fusiform basidiospores, and absence of clamp connections. nrITS‐based phylogenetic analysis places both species in a distinct, well‐supported lineage. Molecular phylogeny resolved C. sulcatipes as a distinct lineage separate from the C. flavocontextus and C. sinopulverulentus clade, and G. binkatensis as an independent clade within G . sect. Castaneus , both strongly supported by bootstrap analyses. Comprehensive descriptions, in situ photographs, and micrographs relative to new species are provided.

Recent collections of Rhodotus from the Midwestern United States, analyzed through genetic studies conducted by Mycota Labs and The Ohio Mushroom DNA Lab, exhibit phylogenetic divergence from the European collections of R. palmatus. Morphological and molecular data indicate that these midwestern collections correspond to a previously described species, Agaricus reticeps, requiring its recombination within Rhodotus.

This extraction protocol uses an Extract-N-Amp equivalent solution to quickly and cheaply perform DNA extractions. This protocol has been widely used for dried specimens of macrofungi, but will work for other organismal groups as well. This protocol is an amalgamation of two different protocols that are currently in use by academic labs in the United States. This protocol is easily scalable for larger batch sizes. The standard protocol assumes 100mL batches. This is enough extraction solution for 5,000 specimens. The primary thing needed to scale up are larger sizes of the sterile vacuum filter flasks. They come in each size you may need - 250mL, 500mL, or 1000mL. For larger batch considerations: 100mL is enough for ~5,000 extractions 250mL is enough for ~12,500 extractions 500mL is enough for ~25,000 extractions 1000mL is enough for ~50,000 extractions Our lab typically make 500mL batches at a time.

We obtained an ITS sequence from the holotype of Volvaria umbonata Peck. DNA data shows that this is the correct name for the species that has been assigned the temporary code Volvariella "sp-IN02". A formal nomenclatural combination in Volvariella is made here.

This protocol is best used when preparing macrofungal specimens for Sanger sequencing or as a secondary extraction protocol for ONT nanopore barcoding. We also utilize it as a primary extraction protocol for working with older museum collections of macrofungi when clean DNA is needed to be retained with the collections. Each step in this protocol can be expanded to work with much larger sets. It is common for us to work with 96 or more specimens at a time.