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1. Liftoff: an accurate gene annotation mapping tool

   https://doi.org/10.1101/2020.06.24.169680  

   Shumate, A. ; Salzberg, S.L. ( June 2020 , bioRxiv)

   Improvements in DNA sequencing technology and computational methods have led to a substantial increase in the creation of high-quality genome assemblies of many species. To understand the biology of these genomes, annotation of gene features and other functional elements is essential; however for most species, only the reference genome is well-annotated. One strategy to annotate new or improved genome assemblies is to map or ‘lift over’ the genes from a previously-annotated reference genome. Here we describe Liftoff, a new genome annotation lift-over tool capable of mapping genes between two assemblies of the same or closely-related species. Liftoff aligns genes from a reference genome to a target genome and finds the mapping that maximizes sequence identity while preserving the structure of each exon, transcript, and gene. We show that Liftoff can accurately map 99.9% of genes between two versions of the human reference genome with an average sequence identity >99.9%. We also show that Liftoff can map genes across species by successfully lifting over 98.4% of human protein-coding genes to a chimpanzee genome assembly with 98.7% sequence identity. Availability The source code for Liftoff is available at https://github.com/agshumate/Liftoff
2. From telomere to telomere: The transcriptional and epigenetic state of human repeat elements

   https://doi.org/10.1126/science.abk3112  

   Hoyt, Savannah J. ; Storer, Jessica M. ; Hartley, Gabrielle A. ; Grady, Patrick G. ; Gershman, Ariel ; de Lima, Leonardo G. ; Limouse, Charles ; Halabian, Reza ; Wojenski, Luke ; Rodriguez, Matias ; et al ( April 2022 , Science)

   INTRODUCTION Transposable elements (TEs), repeat expansions, and repeat-mediated structural rearrangements play key roles in chromosome structure and species evolution, contribute to human genetic variation, and substantially influence human health through copy number variants, structural variants, insertions, deletions, and alterations to gene transcription and splicing. Despite their formative role in genome stability, repetitive regions have been relegated to gaps and collapsed regions in human genome reference GRCh38 owing to the technological limitations during its development. The lack of linear sequence in these regions, particularly in centromeres, resulted in the inability to fully explore the repeat content of the human genome in the context of both local and regional chromosomal environments. RATIONALE Long-read sequencing supported the complete, telomere-to-telomere (T2T) assembly of the pseudo-haploid human cell line CHM13. This resource affords a genome-scale assessment of all human repetitive sequences, including TEs and previously unknown repeats and satellites, both within and outside of gaps and collapsed regions. Additionally, a complete genome enables the opportunity to explore the epigenetic and transcriptional profiles of these elements that are fundamental to our understanding of chromosome structure, function, and evolution. Comparative analyses reveal modes of repeat divergence, evolution, and expansion or contraction with locus-level resolution. RESULTS We implementedmore »a comprehensive repeat annotation workflow using previously known human repeats and de novo repeat modeling followed by manual curation, including assessing overlaps with gene annotations, segmental duplications, tandem repeats, and annotated repeats. Using this method, we developed an updated catalog of human repetitive sequences and refined previous repeat annotations. We discovered 43 previously unknown repeats and repeat variants and characterized 19 complex, composite repetitive structures, which often carry genes, across T2T-CHM13. Using precision nuclear run-on sequencing (PRO-seq) and CpG methylated sites generated from Oxford Nanopore Technologies long-read sequencing data, we assessed RNA polymerase engagement across retroelements genome-wide, revealing correlations between nascent transcription, sequence divergence, CpG density, and methylation. These analyses were extended to evaluate RNA polymerase occupancy for all repeats, including high-density satellite repeats that reside in previously inaccessible centromeric regions of all human chromosomes. Moreover, using both mapping-dependent and mapping-independent approaches across early developmental stages and a complete cell cycle time series, we found that engaged RNA polymerase across satellites is low; in contrast, TE transcription is abundant and serves as a boundary for changes in CpG methylation and centromere substructure. Together, these data reveal the dynamic relationship between transcriptionally active retroelement subclasses and DNA methylation, as well as potential mechanisms for the derivation and evolution of new repeat families and composite elements. Focusing on the emerging T2T-level assembly of the HG002 X chromosome, we reveal that a high level of repeat variation likely exists across the human population, including composite element copy numbers that affect gene copy number. Additionally, we highlight the impact of repeats on the structural diversity of the genome, revealing repeat expansions with extreme copy number differences between humans and primates while also providing high-confidence annotations of retroelement transduction events. CONCLUSION The comprehensive repeat annotations and updated repeat models described herein serve as a resource for expanding the compendium of human genome sequences and reveal the impact of specific repeats on the human genome. In developing this resource, we provide a methodological framework for assessing repeat variation within and between human genomes. The exhaustive assessment of the transcriptional landscape of repeats, at both the genome scale and locally, such as within centromeres, sets the stage for functional studies to disentangle the role transcription plays in the mechanisms essential for genome stability and chromosome segregation. Finally, our work demonstrates the need to increase efforts toward achieving T2T-level assemblies for nonhuman primates and other species to fully understand the complexity and impact of repeat-derived genomic innovations that define primate lineages, including humans. Telomere-to-telomere assembly of CHM13 supports repeat annotations and discoveries. The human reference T2T-CHM13 filled gaps and corrected collapsed regions (triangles) in GRCh38. Combining long read–based methylation calls, PRO-seq, and multilevel computational methods, we provide a compendium of human repeats, define retroelement expression and methylation profiles, and delineate locus-specific sites of nascent transcription genome-wide, including previously inaccessible centromeres. SINE, short interspersed element; SVA, SINE–variable number tandem repeat– Alu ; LINE, long interspersed element; LTR, long terminal repeat; TSS, transcription start site; pA, xxxxxxxxxxxxxxxx.« less
3. Grouper: graph-based clustering and annotation for improved de novo transcriptome analysis

   https://doi.org/10.1093/bioinformatics/bty378  

   Malik, Laraib ; Almodaresi, Fatemeh ; Patro, Rob ; Birol, ed., Inanc ( May 2018 , Bioinformatics)

   De novo transcriptome analysis using RNA-seq offers a promising means to study gene expression in non-model organisms. Yet, the difficulty of transcriptome assembly means that the contigs provided by the assembler often represent a fractured and incomplete view of the transcriptome, complicating downstream analysis. We introduce Grouper, a new method for clustering contigs from de novo assemblies that are likely to belong to the same transcripts and genes; these groups can subsequently be analyzed more robustly. When provided with access to the genome of a related organism, Grouper can transfer annotations to the de novo assembly, further improving the clustering.

   On de novo assemblies from four different species, we show that Grouper is able to accurately cluster a larger number of contigs than the existing state-of-the-art method. The Grouper pipeline is able to map greater than 10% more reads against the contigs, leading to accurate downstream differential expression analyses. The labeling module, in the presence of a closely related annotated genome, can efficiently transfer annotations to the contigs and use this information to further improve clustering. Overall, Grouper provides a complete and efficient pipeline for processing de novo transcriptomic assemblies.

   The Grouper software is freely available atmore »https://github.com/COMBINE-lab/grouper under the 2-clause BSD license.

   Supplementary data are available at Bioinformatics online.

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4. A New Pipeline for Removing Paralogs in Target Enrichment Data

   https://doi.org/10.1093/sysbio/syab044  

   Zhou, Wenbin ; Soghigian, John ; Xiang, Qiu-Yun ( June 2021 , Systematic Biology)

   Abstract Target enrichment (such as Hyb-Seq) is a well-established high throughput sequencing method that has been increasingly used for phylogenomic studies. Unfortunately, current widely used pipelines for analysis of target enrichment data do not have a vigorous procedure to remove paralogs in target enrichment data. In this study, we develop a pipeline we call Putative Paralogs Detection (PPD) to better address putative paralogs from enrichment data. The new pipeline is an add-on to the existing HybPiper pipeline, and the entire pipeline applies criteria in both sequence similarity and heterozygous sites at each locus in the identification of paralogs. Users may adjust the thresholds of sequence identity and heterozygous sites to identify and remove paralogs according to the level of phylogenetic divergence of their group of interest. The new pipeline also removes highly polymorphic sites attributed to errors in sequence assembly and gappy regions in the alignment. We demonstrated the value of the new pipeline using empirical data generated from Hyb-Seq and the Angiosperm 353 kit for two woody genera Castanea (Fagaceae, Fagales) and Hamamelis (Hamamelidaceae, Saxifragales). Comparisons of datasets showed that the PPD identified many more putative paralogs than the popular method HybPiper. Comparisons of tree topologies and divergence timesmore »showed evident differences between data from HybPiper and data from our new PPD pipeline. We further evaluated the accuracy and error rates of PPD by BLAST mapping of putative paralogous and orthologous sequences to a reference genome sequence of Castanea mollissima. Compared to HybPiper alone, PPD identified substantially more paralogous gene sequences that mapped to multiple regions of the reference genome (31 genes for PPD compared with 4 genes for HybPiper alone). In conjunction with HybPiper, paralogous genes identified by both pipelines can be removed resulting in the construction of more robust orthologous gene datasets for phylogenomic and divergence time analyses. Our study demonstrates the value of Hyb-Seq with data derived from the Angiosperm 353 probe set for elucidating species relationships within a genus, and argues for the importance of additional steps to filter paralogous genes and poorly aligned regions (e.g., as occur through assembly errors), such as our new PPD pipeline described in this study.« less
5. Genome Report: Whole Genome Sequence and Annotation of the Parasitoid Jewel Wasp Nasonia giraulti Laboratory Strain RV2X[u]

   https://doi.org/10.1534/g3.120.401200  

   Wang, Xiaozhu ; Kelkar, Yogeshwar D. ; Xiong, Xiao ; Martinson, Ellen O. ; Lynch, Jeremy ; Zhang, Chao ; Werren, John H. ; Wang, Xu ( August 2020 , G3: Genes|Genomes|Genetics)

   Jewel wasps in the genus of Nasonia are parasitoids with haplodiploidy sex determination, rapid development and are easy to culture in the laboratory. They are excellent models for insect genetics, genomics, epigenetics, development, and evolution. Nasonia vitripennis ( Nv ) and N. giraulti ( Ng ) are closely-related species that can be intercrossed, particularly after removal of the intracellular bacterium Wolbachia , which serve as a powerful tool to map and positionally clone morphological, behavioral, expression and methylation phenotypes. The Nv reference genome was assembled using Sanger, PacBio and Nanopore approaches and annotated with extensive RNA-seq data. In contrast, Ng genome is only available through low coverage resequencing. Therefore, de novo Ng assembly is in urgent need to advance this system. In this study, we report a high-quality Ng assembly using 10X Genomics linked-reads with 670X sequencing depth. The current assembly has a genome size of 259,040,977 bp in 3,160 scaffolds with 38.05% G-C and a 98.6% BUSCO completeness score. 97% of the RNA reads are perfectly aligned to the genome, indicating high quality in contiguity and completeness. A total of 14,777 genes are annotated in the Ng genome, and 72% of the annotated genes have a one-to-one ortholog inmore »the Nv genome. We reported 5 million Ng-Nv SNPs which will facility mapping and population genomic studies in Nasonia . In addition, 42 Ng -specific genes were identified by comparing with Nv genome and annotation. This is the first de novo assembly for this important species in the Nasonia model system, providing a useful new genomic toolkit.« less