Era of gapless plant genomes: innovations in sequencing and mapping technologies revolutionize genomics and breeding

Whole-genome sequencing and assembly have revolutionized plant genetics and molecular biology over the last two decades. However, significant shortcomings in first- and second-generation technology resulted in imperfect reference genomes: numerous and large gaps of low quality or undeterminable sequence in areas of highly repetitive DNA along with limited chromosomal phasing restricted the ability of researchers to characterize regulatory noncoding elements and genic regions that underwent recent duplication events. Recently, advances in long-read sequencing have resulted in the first gapless, telomere-to-telomere (T2T) assemblies of plant genomes. This leap forward has the potential to increase the speed and confidence of genomics and molecular experimentation while reducing costs for the research community

Detailed Analysis of a Contiguous 22-Mb Region of the Maize Genome

Most of our understanding of plant genome structure and evolution has come from the careful annotation of small (e.g., 100 kb) sequenced genomic regions or from automated annotation of complete genome sequences. Here, we sequenced and carefully annotated a contiguous 22 Mb region of maize chromosome 4 using an improved pseudomolecule for annotation. The sequence segment was comprehensively ordered, oriented, and confirmed using the maize optical map. Nearly 84% of the sequence is composed of transposable elements (TEs) that are mostly nested within each other, of which most families are low-copy. We identified 544 gene models using multiple levels of evidence, as well as five miRNA genes. Gene fragments, many captured by TEs, are prevalent within this region. Elimination of gene redundancy from a tetraploid maize ancestor that originated a few million years ago is responsible in this region for most disruptions of synteny with sorghum and rice. Consistent with other sub-genomic analyses in maize, small RNA mapping showed that many small RNAs match TEs and that most TEs match small RNAs. These results, performed on ∼1% of the maize genome, demonstrate the feasibility of refining the B73 RefGen_v1 genome assembly by incorporating optical map, high-resolution genetic map, and comparative genomic data sets. Such improvements, along with those of gene and repeat annotation, will serve to promote future functional genomic and phylogenomic research in maize and other grasses

Genetic and Physical Characterization of Tn904.

The possibility that a streptomycin resistance determinant was part of a transposon was confirmed. Genetic techniques were used to demonstrate definitively that the genetic element, designated Tn904, was capable of transposition in a recA('-) genetic background. Physical analysis of plasmids that had acquired Tn904 showed that in all cases acquisition of the transposon was accompanied by the acquisition of a discrete DNA segment. Transposition was not site-specific with respect to the recipient molecule. It was shown at the level of resistance to streptomycin conferred by Tn904 was directly related to the copy number of the transposon. As a result, recovery of cells in which transposition of Tn904 from a low copy number plasmid to a high copy number plasmid had occurred could be enriched for by growth of the cells on Sm containing media. The practical implications of this phenomenon were discussed. Restriction endonuclease digest analysis of Tn904 showed that the transposon had a size of 5.2 kb. Electron microscopic heteroduplex analysis showed that the transposon was flanked by inverted repeats of about 125 bp. A coordinate system based on this data was devised for Tn904. The approximate site of the Sm resistance determinant was localized on the transposon. The transposon was shown not to increase in size when exposed to high levels of streptomycin. The transposon was shown not to include markers for mercury or spectinomycin resistance. Transposition frequency in two different experimental situations was determined. Precise excision of Tn904 from plasmid molecules was not observed to occur.Ph.D.GeneticsUniversity of Michiganhttp://deepblue.lib.umich.edu/bitstream/2027.42/159047/1/8225007.pd

Atypical Creutzfeldt-Jakob Disease in an American Family with an Insert Mutation in the PRNP Amyloid Precursor Gene.

An American family of English origin with an unusually early onset and long-duration form of Creutzfeldt-Jakob disease (CJD) had a heterozygous insert mutation in the region of repeating octapeptide coding sequences between codons 51 and 91 of the PRNP gene on chromosome 20. Affected members were 23 to 35 years old at the onset of illnesses that lasted from 4 to 13 years, yet experimental transmission of disease from the proband (11-year duration) produced a typically brief incubation period and duration of illness in each of three inoculated primates. Also, the PrP amyloid protein that accumulates in CJD brain was only barely detectable in extracted brain tissue from one case with massive spongiform change and was undetectable in another case with no spongiform change, perhaps because of epitope shielding by a configurational change in the protein induced by the mutation. Analysis of this and other families with similar inserts suggests that such mutations in the PRNP gene not only predispose to CJD, but also modify its phenotypic expression

Familial long-read sequencing increases yield of de novo mutations

Studies of de novo mutation (DNM) have typically excluded some of the most repetitive and complex regions of the genome because these regions cannot be unambiguously mapped with short-read sequencing data. To better understand the genome-wide pattern of DNM, we generated long-read sequence data from an autism parent-child quad with an affected female where no pathogenic variant had been discovered in short-read Illumina sequence data. We deeply sequenced all four individuals by using three sequencing platforms (Illumina, Oxford Nanopore, and Pacific Biosciences) and three complementary technologies (Strand-seq, optical mapping, and 10X Genomics). Using long-read sequencing, we initially discovered and validated 171 DNMs across two children-a 20% increase in the number of de novo single-nucleotide variants (SNVs) and indels when compared to short-read callsets. The number of DNMs further increased by 5% when considering a more complete human reference (T2T-CHM13) because of the recovery of events in regions absent from GRCh38 (e.g., three DNMs in heterochromatic satellites). In total, we validated 195 de novo germline mutations and 23 potential post-zygotic mosaic mutations across both children; the overall true substitution rate based on this integrated callset is at least 1.41 × 10-8 substitutions per nucleotide per generation. We also identified six de novo insertions and deletions in tandem repeats, two of which represent structural variants. We demonstrate that long-read sequencing and assembly, especially when combined with a more complete reference genome, increases the number of DNMs by >25% compared to previous studies, providing a more complete catalog of DNM compared to short-read data alone

Prepublication data sharing.

Rapid release of prepublication data has served the field of genomics well. Attendees at a workshop in Toronto recommend extending the practice to other biological data sets