De novo PacBio long-read and phased avian genome assemblies correct and add to reference genes generated with intermediate and short reads

Abstract

Reference-quality genomes are expected to provide a resource for studying gene structure, function, and evolution. However, often genes of interest are not completely or accurately assembled, leading to unknown errors in analyses or additional cloning efforts for the correct sequences. A promising solution is long-read sequencing. Here we tested PacBio-based long-read sequencing and diploid assembly for potential improvements to the Sanger-based intermediate-read zebra finch reference and Illumina-based short-read Anna's hummingbird reference, 2 vocal learning avian species widely studied in neuroscience and genomics. With DNA of the same individuals used to generate the reference genomes, we generated diploid assemblies with the FALCON-Unzip assembler, resulting in contigs with no gaps in the megabase range, representing 150-fold and 200-fold improvements over the current zebra finch and hummingbird references, respectively. These long-read and phased assemblies corrected and resolved what we discovered to be numerous misassemblies in the references, including missing sequences in gaps, erroneous sequences flanking gaps, base call errors in difficult-to-sequence regions, complex repeat structure errors, and allelic differences between the 2 haplotypes. These improvements were validated by single long-genome and transcriptome reads and resulted for the first time in completely resolved protein-coding genes widely studied in neuroscience and specialized in vocal learning species. These findings demonstrate the impact of long reads, sequencing of previously difficult-to-sequence regions, and phasing of haplotypes on generating the high-quality assemblies necessary for understanding gene structure, function, and evolution.

High-resolution comparative analysis of great ape genomes

“…Our great ape genome assemblies improved sequence contiguity by orders of magnitude (20, 60), leading to a more comprehensive understanding of the evolution of structural variation. Coupling this effort with full-length cDNA sequencing improved gene annotation, especially for the discovery of new transcripts and isoforms that have recently diverged between closely related species.…”

The Juicebox Assembly Tools module facilitatesde novoassembly of mammalian genomes with chromosome-length scaffolds for under $1000

“…Scaffolds can contain errors in contig order (a 'translocation') or orientation (an 'inversion'). Examples of such errors can be found in the best available reference genomes for many species (Robert B. Norgren 2013;Shearer et al 2014;Tang et al 2014;Chen et al 2015;Davey et al 2016;Utsunomiya et al 2016;Schneider et al 2017;Korlach et al 2017). Consequently, inexpensive methods for identifying and correcting assembly errors are crucial for the generation of accurate assemblies (Salzberg and Yorke 2005;Phillippy, Schatz, and Pop 2008;Gnerre et al 2009;Tsai, Otto, and Berriman 2010;Salzberg et al 2012;Hunt et al 2013;Gurevich et al 2013;Bradnam et al 2013;Simão et al 2015;Fierst 2015;Muggli et al 2015;Yuan et al 2017;Harewood et al 2017).…”

Comparative Annotation Toolkit (CAT) - simultaneous clade and personal genome annotation

“…Much effort has been put into reducing these errors, including the introduction of diploid assembly methods [80], which reduce the rate of indel errors in the assembly caused by collapsed heterozygosity. However, our analysis of recent diploid assemblies produced by the PacBio tool Falcon Unzip [80] suggests that indel errors are still problematic and inflate the number of frame-shifting indels, with around 1% of transcripts frame-shifted in a diploid Zebrafinch assembly [81] compared to the Illumina based assembly of the same species. Additionally, these errors appear in assemblies of haploid cell lines, suggesting that heterozygosity is not the only cause.…”

“…both Pacific Biosciences [80,144,81] and 10x Genomics [145] provide tools to construct phased, diploid assemblies. Annotating diploid assemblies provides a window into haplotype-specific structural variation that may affect gene expression.…”