PURC v2.0: a program for improved sequence inference for polyploid phylogenetics and other manifestations of the multiple-copy problem

Inferring the true biological sequences from amplicon mixtures remains a difficult bioinformatic problem. The traditional approach is to cluster sequencing reads by similarity thresholds and treat the consensus sequence of each cluster as an "operational taxonomic unit" (OTU). Recently, this approach has been improved upon by model-based methods that correct PCR and sequencing errors in order to infer "amplicon sequence variants" (ASVs). To date, ASV approaches have been used primarily in metagenomics, but they are also useful for identifying allelic or paralogous variants and for determining homeologs in polyploid organisms. To facilitate the usage of ASV methods among polyploidy researchers, we incorporated ASV inference alongside OTU clustering in PURC v2.0, a major update to PURC (Pipeline for Untangling Reticulate Complexes). In addition to preserving original PURC functions, PURC v2.0 allows users to process PacBio CCS/HiFi reads through DADA2 to generate and annotate ASVs for multiplexed data, with outputs including separate alignments for each locus ready for phylogenetic inference. In addition, PURC v2.0 features faster demultiplexing than the original version and has been updated to be compatible with Python 3. In this chapter we present results indicating that PURC v2.0 (using the ASV approach) is more likely to infer the correct biological sequences in comparison to the earlier OTU-based PURC, and describe how to prepare sequencing data, run PURC v2.0 under several different modes, and interpret the output. We expect that PURC v2.0 will provide biologists with a method for generating multi-locus "moderate data" datasets that are large enough to be phylogenetically informative and small enough for manual curation.

Predicate fronting with verb doubling in Krachi: A parallel chains analysis

This article examines verb doubling predicate focus constructions in Krachi, an endangered language of Ghana. Krachi has three such constructions: one where V alone appears in the left periphery; another where VO has been fronted; and a third involving OV inversion in the fronted constituent. Regardless of the fronted expression, the constructions can be interpreted either contrastively or exhaustively. We argue that all three constructions involve the same mechanism – the formation of parallel chains anchored to the same syntactic object. We propose that the parallel chains formed in all three cases are identical, involving one v0-to-T0 head movement chain and one v’-to-Spec, FocP A-bar chain. The reduction of these chains at PF yields the surface doubling of the predicate without appeal to multiple copy spell-out. We propose that minor differences in the PF interpretation of the peripheral v’ copy account for the differences in word order between the three constructions.

Predicate fronting in Yiddish and conditions on multiple copy Spell-Out

Predicate fronting with doubling (also known as the predicate cleft) has long been a challenge for theories of syntax that do not predict the pronunciation of multiple occurrences. Previous analyses that derive the construction via syntactic movement, including those attributing verb doubling to the formation of parallel chains (e.g., Aboh 2006; Kandybowicz 2008), are incompatible with remnant movement (Müller 1998), which does not give rise to doubling. This article presents data from the predicate fronting construction in Yiddish, in which verbs always double but complements never do. I argue that these seemingly contradictory pronunciation facts can be reconciled even if one assumes that phrasal movement and head movement are both syntactic. More specifically, the pronunciation of occurrences in Yiddish (doubled or not) follows from the general conditions on Spell-Out (or Transferpf) defined by Collins and Stabler (2016), modified only to accommodate syntactic head movement. Post-syntactic PF repairs are thus not required to account for the facts of the Yiddish predicate fronting construction. If such repairs are needed to generate doubling phenomena in other languages, they should be explicitly defined so as to modify or override the predictions of default Spell-Out conditions.

Precise and broad scope genome editing based on high-specificity Cas9 nickases

RNA-guided nucleases (RGNs) based on CRISPR systems permit installing short and large edits within eukaryotic genomes. However, precise genome editing is often hindered due to nuclease off-target activities and the multiple-copy character of the vast majority of chromosomal sequences. Dual nicking RGNs and high-specificity RGNs both exhibit low off-target activities. Here, we report that high-specificity Cas9 nucleases are convertible into nicking Cas9D10A variants whose precision is superior to that of the commonly used Cas9D10A nickase. Dual nicking RGNs based on a selected group of these Cas9D10A variants can yield gene knockouts and gene knock-ins at frequencies similar to or higher than those achieved by their conventional counterparts. Moreover, high-specificity dual nicking RGNs are capable of distinguishing highly similar sequences by ‘tiptoeing’ over pre-existing single base-pair polymorphisms. Finally, high-specificity RNA-guided nicking complexes generally preserve genomic integrity, as demonstrated by unbiased genome-wide high-throughput sequencing assays. Thus, in addition to substantially enlarging the Cas9 nickase toolkit, we demonstrate the feasibility in expanding the range and precision of DNA knockout and knock-in procedures. The herein introduced tools and multi-tier high-specificity genome editing strategies might be particularly beneficial whenever predictability and/or safety of genetic manipulations are paramount.

Musical Classicisms in Italy Before the Madrigal

Modern evaluations of the relation between music and the fashion for the antique in Italy in the period before the madrigal have tended to proceed from the perspective of intellectual history. This article aims to offer an alternative—although certainly related—perspective, by exploring the circulation of musical classicisms in Italian visual and material culture, roughly from 1450 to 1520. This period saw the rise to prominence in Italy of both commercial text printing and other multiple-copy formats such as the art print, the medal, the bronze plaquette, and a little later historiated maiolica. These technologies offer a particularly compelling lens through which to examine musical encounters with classical antiquity that were not motivated by an expert professional interest in either music or classical texts, but rather characterized by an investment in antiquity as a fashionable source of cultural capital.