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

Fiddes, Ian T.; Armstrong, Joel; Diekhans, Mark; Nachtweide, Stefanie; Kronenberg, Zev; Underwood, Jason G.; Gordon, David; Earl, Dent; Keane, Thomas M.; Eichler, Evan E.; Haussler, David; Stanke, Mario; Paten, Benedict

doi:10.1101/gr.233460.117

Cited by 104 publications

(102 citation statements)

References 60 publications

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“…Comparative Annotation Toolkit (CAT) is a software pipeline that leverages whole-genome alignments, existing annotations, and comparative gene prediction tools to simultaneously annotate multiple genomes, defining orthologous relationships and discovering gene family expansion and contraction 27 . CAT also diagnoses assembly quality by investigating the rate of gene model-breaking indels seen in transcript projections from a reference, as well as looking at the rate of transcript projections that map in a disjointed fashion.…”

Section: Resultsmentioning

confidence: 99%

“…In every place where the alignment indicated a difference in order and orientation of scaffolds between the two assemblies, we used every available data type to resolve the discrepancy and determine which was correct. Our strategies included aligning BAC-end pairs from a half-brother of Twilight 2 to the assemblies using bwa mem with default parameters 34 , assessing concordance with the physical map, looking for split genes predicted by the CAT 27 , aligning coding sequences of any genes in the region to the assemblies using gmap with default parameters 35 , and examining heatmaps of long-range read pairs mapping to the assembly generated by the HiRise and longranger pipelines 24 .…”

Section: Methodsmentioning

confidence: 99%

“…The guide tree was (((Human:0.164501,((Pig:0.12,Cow:0.16908)1:0.02,(EquCab3:0.0001,EquCab2:0.0001):0.059397,White_rhinoceros:0.05)1:0.060727)1:0.032898)1:0.023664,Elephant:0.155646), putting EquCab2 and EquCab3 under the same node with a branch length of 0.0001. CAT 27 was then run using the Ensembl V89 annotation of pig as the source transcript set. No RNA-seq data were provided, so no transcript cleanup steps or comparative gene predictions were performed.…”

Section: Methodsmentioning

confidence: 99%

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Improved reference genome for the domestic horse increases assembly contiguity and composition

et al. 2018

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Recent advances in genomic sequencing technology and computational assembly methods have allowed scientists to improve reference genome assemblies in terms of contiguity and composition. EquCab2, a reference genome for the domestic horse, was released in 2007. Although of equal or better quality compared to other first-generation Sanger assemblies, it had many of the shortcomings common to them. In 2014, the equine genomics research community began a project to improve the reference sequence for the horse, building upon the solid foundation of EquCab2 and incorporating new short-read data, long-read data, and proximity ligation data. Here, we present EquCab3. The count of non-N bases in the incorporated chromosomes is improved from 2.33 Gb in EquCab2 to 2.41 Gb in EquCab3. Contiguity has also been improved nearly 40-fold with a contig N50 of 4.5 Mb and scaffold contiguity enhanced to where all but one of the 32 chromosomes is comprised of a single scaffold.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Improved reference genome for the domestic horse increases assembly contiguity and composition

et al. 2018

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“…( Table 1, Supplementary Note 4). Comparative annotation of our whole-genome assembly also shows a higher agreement of mapped transcripts than previous assemblies and only a slightly increased rate of potential frameshifts compared to GRCh38 23 . Of the 19,618 protein-coding genes annotated in the CHM13 de novo assembly, just 170 (0.86%) contain a predicted frameshift, or, if measured by transcripts, only 334 of 83,332 transcripts (0.40%) contain a predicted frameshift (Supplementary Table 1).…”

Section: Highly Continuous Whole-genome Assemblymentioning

confidence: 65%

Telomere-to-telomere assembly of a complete human X chromosome

Miga

Koren

Rhie

et al. 2020

Nature

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After two decades of improvements, the current human reference genome (GRCh38) is the most accurate and complete vertebrate genome ever produced. However, no single chromosome has been finished end to end, and hundreds of unresolved gaps persist 1,2. Here we present a human genome assembly that surpasses the continuity of GRCh38 2 , along with a gapless, telomere-to-telomere assembly of a human chromosome. This was enabled by high-coverage, ultra-long-read nanopore sequencing of the complete hydatidiform mole CHM13 genome, combined with complementary technologies for quality improvement and validation. Focusing our efforts on the human X chromosome 3 , we reconstructed the centromeric satellite DNA array (approximately 3.1 Mb) and closed the 29 remaining gaps in the current reference, including new sequences from the human pseudoautosomal regions and from cancer-testis ampliconic gene families (CT-X and GAGE). These sequences will be integrated into future human reference genome releases. In addition, the complete chromosome X, combined with the ultra-long nanopore data, allowed us to map methylation patterns across complex tandem repeats and satellite arrays. Our results demonstrate that finishing the entire human genome is now within reach, and the data presented here will facilitate ongoing efforts to complete the other human chromosomes. Complete, telomere-to-telomere reference genome assemblies are necessary to ensure that all genomic variants are discovered and studied. At present, unresolved areas of the human genome are defined by multi-megabase satellite arrays in the pericentromeric regions and the ribosomal DNA arrays on acrocentric short arms, as well as regions enriched in segmental duplications that are greater than hundreds of kilobases in length and that exhibit sequence identity of more than 98% between paralogues. Owing to their absence from the reference, these repeat-rich sequences are often excluded from genetics and genomics studies, which limits the scope of association and functional analyses 4,5. Unresolved repeat sequences also result in unintended consequences; for example, paralogous sequence variants incorrectly being called as allelic variants 6 , and the contamination of bacterial gene databases 7. Completion of the entire human genome is expected to contribute to our understanding of chromosome function 8 , human disease 9 and genomic variation, which will improve technologies in biomedicine that use short-read mapping to a reference genome (for example, RNA sequencing (RNA-seq) 10 , chromatin immunoprecipitation followed by sequencing (ChIP-seq) 11 and assay for transposase-accessible chromatin using sequencing (ATAC-seq) 12). The fundamental challenge of reconstructing a genome from many comparatively short sequencing reads-a process known as genome assembly-is distinguishing the repeated sequences from one another 13. Resolving such repeats relies on sequencing reads that are long enough to span the entire repeat or accurate enough to distinguish each repeat copy on the basis of...

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“…We ran the Comparative Annotation Toolkit 52 to annotate the polished assemblies in order to analyze how well Shasta assembles transcripts and genes. Each assembly was individually aligned to the GRCh38 reference assembly using Cactus 53 to create the input alignment to CAT.…”

Section: Analysis Methodsmentioning

confidence: 99%

Nanopore sequencing and the Shasta toolkit enable efficient de novo assembly of eleven human genomes

et al. 2020

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De novo assembly of a human genome using nanopore long-read sequences has been reported but it used more than 150,000 CPU hours and weeks of wall-clock time. To enable rapid human genome assembly we present Shasta, a de novo long read assembler, and polishing algorithms named MarginPolish and HELEN. Using a single PromethION nanopore sequencer and our toolkit, we assembled eleven highly contiguous human genomes de novo in nine days. We achieved ~63x coverage, 42 Kb read N50, and 6.5x coverage in 100 Kb+ reads using three flow cells per sample. Shasta produced a complete haploid human genome assembly in under six hours on a single commercial compute node. MarginPolish and HELEN polished haploid assemblies to more than 99.9% identity (QV30) with nanopore reads alone. Addition of proximity ligation (Hi-C) sequencing enabled near chromosome-level scaffolds for all eleven genomes. We compare our assembly performance to existing methods for diploid, haploid, and trio-binned human samples and report superior accuracy and speed.

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

Cited by 104 publications

References 60 publications

Improved reference genome for the domestic horse increases assembly contiguity and composition

Improved reference genome for the domestic horse increases assembly contiguity and composition

Telomere-to-telomere assembly of a complete human X chromosome

Nanopore sequencing and the Shasta toolkit enable efficient de novo assembly of eleven human genomes

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