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High-throughput short-read sequencing has taken on a central role in research and diagnostics. Hundreds of different assays take advantage of Illumina short-read sequencers, the predominant short-read sequencing technology available today. Although other short-read sequencing technologies exist, the ubiquity of Illumina sequencers in sequencing core facilities and the high capital costs of these technologies have limited their adoption. Among a new generation of sequencing technologies, Oxford Nanopore Technologies (ONT) holds a unique position because the ONT MinION, an error-prone long-read sequencer, is associated with little to no capital cost. Here we show that we can make short-read Illumina libraries compatible with the ONT MinION by using the rolling circle to concatemeric consensus (R2C2) method to circularize and amplify the short library molecules. This results in longer DNA molecules containing tandem repeats of the original short library molecules. This longer DNA is ideally suited for the ONT MinION, and after sequencing, the tandem repeats in the resulting raw reads can be converted into high-accuracy consensus reads with similar error rates to that of the Illumina MiSeq. We highlight this capability by producing and benchmarking RNA-seq, ChIP-seq, and regular and target-enriched Tn5 libraries. We also explore the use of this approach for rapid evaluation of sequencing library metrics by implementing a real-time analysis workflow. 

Abstract Promoters and the noncoding sequences that drive their function are fundamental aspects of genes that are critical to their regulation. The transcription preinitiation complex binds and assembles on promoters where it facilitates transcription. The transcription start site (TSS) is located downstream of the promoter sequence and is defined as the location in the genome where polymerase begins transcribing DNA into RNA. Knowing the location of TSSs is useful for annotation of genes, identification of non‐coding sequences important to gene regulation, detection of alternative TSSs, and understanding of 5′ UTR content. Several existing techniques make it possible to accurately identify TSSs, but are often difficult to perform experimentally, require large amounts of input RNA, or are unable to identify a large number of TSSs from a single sample. Many of these protocols take advantage of template switching reverse transcriptases (TSRTs), which reliably place an adaptor at the 5′ end of a first strand synthesis of cDNA. Here, we introduce a protocol that exploits TSRT activity combined with rolling circle amplification to identify TSSs with several unique advantages over existing methods. Sequence adaptors are placed on the 5′ and 3′ end of the full‐length cDNA copy of a transcript. A splint compatible with those adaptors is then used to circularize the full‐length cDNA. Linear DNA containing concatemers of the cDNA are generated using rolling circle amplification, and a sequencing library is formed by fragmenting the concatemers. This protocol is straightforward to execute, requiring limited bench time with relatively stable reagents. Using extremely low amounts of RNA input, this protocol produces large numbers of accurate, deduplicated TSSs genome wide. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Splint generation Basic Protocol 2: RNA extraction Basic Protocol 3: cDNA synthesis Basic Protocol 4: cDNA circularization and amplification Basic Protocol 5: Library generation 

Abstract The Javan gibbon, Hylobates moloch, is an endangered gibbon species restricted to the forest remnants of western and central Java, Indonesia, and one of the rarest of the Hylobatidae family. Hylobatids consist of 4 genera (Holoock, Hylobates, Symphalangus, and Nomascus) that are characterized by different numbers of chromosomes, ranging from 38 to 52. The underlying cause of this karyotype plasticity is not entirely understood, at least in part, due to the limited availability of genomic data. Here we present the first scaffold-level assembly for H. moloch using a combination of whole-genome Illumina short reads, 10X Chromium linked reads, PacBio, and Oxford Nanopore long reads and proximity-ligation data. This Hylobates genome represents a valuable new resource for comparative genomics studies in primates. 

A newly described population of polar bears in southeastern Greenland suggests the potential for climate refugia. 

Abstract The Andean bear is the only extant member of the Tremarctine subfamily and the only extant ursid species to inhabit South America. Here, we present an annotated de novo assembly of a nuclear genome from a captive-born female Andean bear, Mischief, generated using a combination of short and long DNA and RNA reads. Our final assembly has a length of 2.23 Gb, and a scaffold N50 of 21.12 Mb, contig N50 of 23.5 kb, and BUSCO score of 88%. The Andean bear genome will be a useful resource for exploring the complex phylogenetic history of extinct and extant bear species and for future population genetics studies of Andean bears. 

The evolutionarily conserved splicing regulator neuro-oncological ventral antigen 1 (NOVA1) plays a key role in neural development and function.NOVA1also includes a protein-coding difference between the modern human genome and Neanderthal and Denisovan genomes. To investigate the functional importance of an amino acid change in humans, we reintroduced the archaic allele into human induced pluripotent cells using genome editing and then followed their neural development through cortical organoids. This modification promoted slower development and higher surface complexity in cortical organoids with the archaic version ofNOVA1. Moreover, levels of synaptic markers and synaptic protein coassociations correlated with altered electrophysiological properties in organoids expressing the archaic variant. Our results suggest that the human-specific substitution inNOVA1, which is exclusive to modern humans since divergence from Neanderthals, may have had functional consequences for our species’ evolution.