Transcriptional fates of human-specific segmental duplications in brain

Abstract

Despite the importance of duplicate genes for evolutionary adaptation, accurate gene annotation is often incomplete, incorrect, or lacking in regions of segmental duplication. We developed an approach combining long-read sequencing and hybridization capture to yield full-length transcript information and confidently distinguish between nearly identical genes/paralogs. We used biotinylated probes to enrich for full-length cDNA from duplicated regions, which were then amplified, size-fractionated, and sequenced using single-molecule, long-read sequencing technology, permitting us to distinguish between highly identical genes by virtue of multiple paralogous sequence variants. We examined 19 gene families as expressed in developing and adult human brain, selected for their high sequence identity (average >99%) and overlap with human-specific segmental duplications (SDs). We characterized the transcriptional differences between related paralogs to better understand the birth-death process of duplicate genes and particularly how the process leads to gene innovation. In 48% of the cases, we find that the expressed duplicates have changed substantially from their ancestral models due to novel sites of transcription initiation, splicing, and polyadenylation, as well as fusion transcripts that connect duplication-derived exons with neighboring genes. We detect unannotated open reading frames in genes currently annotated as pseudogenes, while relegating other duplicates to nonfunctional status. Our method significantly improves gene annotation, specifically defining full-length transcripts, isoforms, and open reading frames for new genes in highly identical SDs. The approach will be more broadly applicable to genes in structurally complex regions of other genomes where the duplication process creates novel genes important for adaptive traits.

Longest protein, longest transcript or most expression, for accurate gene reconstruction of transcriptomes?

“…De novo gene assembly methods that classify loci have similar discrepancies, as RNA-seq reads are often identical among paralogs. A recent report with methods developed to disentangle artifact from biology finds biological evidence of cross-paralog sharing of coding exons (Dougherty et al 2018), in exception to the common definition, but not a surprise to this author with experience trying to disentangle tandem duplicate genes in a range of species.…”

“…The range of biological complexity and variation found in alternate transcripts and paralogs is high, and potential for reconstruction artifacts is similarly high, in part because both natural and artifactual causes produce the same types transcripts (eg. Dougherty et al 2018 for cross-spliced paralogs). Long-read, single molecule sequencing methods have demonstrated value at recovering accurate transcripts, but as Morillon & Gautheret (2019) indicate, cost-effective and accurate short-read RNA data is an abundant resource for improved computational reconstruction of transcripts.…”

The structure, function, and evolution of a complete human chromosome 8

“…Iso-Seq data generation and sequence analyses RNA was purified from approximately 1 x 10 7 CHM13 cells using an RNeasy kit (Qiagen; 74104) and prepared into Iso-Seq libraries following a standard protocol 68 . Libraries were loaded on two SMRT Cells 8M and sequenced on the Sequel II.…”

The structure, function, and evolution of a complete human chromosome 8

“…Nanopore_methylation_utilities integrates methylation information into the BAM file for viewing in IGV's 67 bisulfite mode, which was used to visualize CpG methylation. Iso-Seq data generation and sequence analyses RNA was purified from approximately 1 x 10 7 CHM13 cells using an RNeasy kit (Qiagen; 74104) and prepared into Iso-Seq libraries following a standard protocol 68 . Libraries were loaded on two SMRT Cells 8M and sequenced on the Sequel II.…”