Mononucleotide repeat expansions with non-natural polymerase substrates

Replicative strand slippage is a biological phenomenon, ubiquitous among different organisms. However, slippage events are also relevant to non-natural replication models utilizing synthetic polymerase substrates. Strand slippage may notably affect the outcome of the primer extension reaction with repetitive templates in the presence of non-natural nucleoside triphosphates. In the current paper, we studied the ability of Taq, Vent (exo-), and Deep Vent (exo-) polymerases to produce truncated, full size, or expanded modified strands utilizing non-natural 2′-deoxyuridine nucleotide analogues and different variants of the homopolymer template. Our data suggest that the slippage of the primer strand is dependent on the duplex fluttering, incorporation efficiency for a particular polymerase-dNTP pair, rate of non-templated base addition, and presence of competing nucleotides.

Genome survey sequencing of Atractylodes lancea and identification of its SSR markers

Atractylodes lancea (Thunb.) DC. is a traditional Chinese medicine rich in sesquiterpenes that has been widely used in China and Japan for the treatment of viral infections. Despite its important pharmacological value, genomic information regarding A. lancea is currently unavailable. In the present study, the whole genome sequence of A. lancea was obtained using an Illumina sequencing platform. The results revealed an estimated genome size for A. lancea of 4,159.24 Mb, with 2.28% heterozygosity, and a repeat rate of 89.2%, all of which indicate a highly heterozygous genome. Based on the genomic data of A. lancea, 27,582 simple sequence repeat (SSR) markers were identified. The differences in representation among nucleotide repeat types were large, e.g., the mononucleotide repeat type was the most abundant (54.74%) while the pentanucleotide repeats were the least abundant (0.10%), and sequence motifs GA/TC (31.17%) and TTC/GAA (7.23%) were the most abundant among the dinucleotide and trinucleotide repeat motifs, respectively. A total of 93,434 genes matched known genes in common databases including 48,493 genes in the Gene Ontology (GO) database and 34,929 genes in the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. This is the first report to sequence and characterize the whole genome of A. lancea and will provide a theoretical basis and reference for further genome-wide deep sequencing and SSR molecular marker development of A. lancea.

Characteristics of induced mutations in offspring derived from irradiated mouse spermatogonia and mature oocytes

The exposure of germ cells to radiation introduces mutations in the genomes of offspring, and a previous whole-genome sequencing study indicated that the irradiation of mouse sperm induces insertions/deletions (indels) and multisite mutations (clustered single nucleotide variants and indels). However, the current knowledge on the mutation spectra is limited, and the effects of radiation exposure on germ cells at stages other than the sperm stage remain unknown. Here, we performed whole-genome sequencing experiments to investigate the exposure of spermatogonia and mature oocytes. We compared de novo mutations in a total of 24 F1 mice conceived before and after the irradiation of their parents. The results indicated that radiation exposure, 4 Gy of gamma rays, induced 9.6 indels and 2.5 multisite mutations in spermatogonia and 4.7 indels and 3.1 multisite mutations in mature oocytes in the autosomal regions of each F1 individual. Notably, we found two types of deletions, namely, small deletions (mainly 1~12 nucleotides) in non-repeat sequences, many of which showed microhomology at the breakpoint junction, and single-nucleotide deletions in mononucleotide repeat sequences. The results suggest that these deletions and multisite mutations could be a typical signature of mutations induced by parental irradiation in mammals.

Intergeneric Relationships within the Family Salicaceae s.l. based on Plastid Phylogenomics

Many Salicaceae s.l. plants are recognized for their important role in the production of products such as wood, oils, and medicines, and as a model organism in life studies. However, the difference in plastid sequence, phylogenetic relationships, and lineage diversification of the family Salicaceae s.l. remain poorly understood. In this study, we compare 24 species representing 18 genera of the family. Simple sequence repeats (SSRs) are considered effective molecular markers for plant species identification and population genetics. Among them, a total of 1798 SSRs were identified, among which mononucleotide repeat was the most common with 1455 accounts representing 80.92% of the total. Most of the SSRs are located in the non-coding region. We also identified five other types of repeats, including 1750 tandems, 434 forward, 407 palindromic, 86 reverse, and 30 complementary repeats. The species in Salicaceae s.l. have a conserved plastid genome. Each plastome presented a typical quadripartite structure and varied in size due to the expansion and contraction of the inverted repeat (IR) boundary, lacking major structural variations, but we identified six divergence hotspot regions. We obtained phylogenetic relationships of 18 genera in Salicaceae s.l. and the 24 species formed a highly supported lineage. Casearia was identified as the basal clade. The divergence time between Salicaceae s.l. and the outgroup was estimated as ~93 Mya; Salix, and Populus diverged around 34 Mya, consistent with the previously reported time. Our research will contribute to a better understanding of the phylogenetic relationships among the members of the Salicaceae s.l.

The complete plastome of real yellow wood (Podocarpus latifolius): gene organization and comparison with related species

Podocarpus latifolius[(Thunb.) R.Br.exMirb.], also known as real yellow wood, is a large evergreen tree with exceptionally high-quality wood. It is a member of the Podocarpaceae family, which includes many species widely grown for wood pulp as well as timber for construction. Despite its importance, studies focusing on its genetic characterization and molecular biology are limited. Therefore, this study reports the complete plastome ofP. latifolius, which is a circular molecule of 134 020 base pairs (bp) in length, lacking a quadripartite structure. TheP. latifoliusplastome encodes 117 unique genes, consisting of 82 protein-coding genes, 31 transfer RNA genes and four ribosomal RNA genes. The analysis showed that the Podocarpaceae plastomes have experienced some intron and gene losses, inversions, and inverted repeat (IR) loss resulting in a diverse plastome organization at the species and genus levels. Therefore, to understand the extent of these genomic rearrangements, more sampling of the Podocarpaceae plastomes is necessary. A total of 149 editing sites were predicted in 28 genes, all of which were C to U conversions. Moreover, a total of 164 simple sequence repeats (SSRs) were identified in theP. latifoliusplastome, the majority being mononucleotide repeat motifs with A/T sequence predominance. Overall, the data obtained in this study will be useful for population genetics, evolutionary history and phylogenetic studies of the species in this genus.

The mutational landscape of MSI-H and MSS colorectal cancer.

e15122 Background: The microsatellite instability-high (MSI-H) phenotype confers good prognosis and greater response to immunotherapy in colorectal cancer(CRC). The mutational landscape of MSI-H CRC is unclear. This study was designed to illustrate the difference mutation profile between the MSI-H and microsatellite stable (MSS) CRC. Methods: Tumor tissue and matched blood samples from 40 patients with colorectal cancer were collected. Microsatellite instability (MSI) status were detected by PCR-amplified for five mononucleotide repeat markers (BAT-25, BAT-26, NR-21, NR-24 and MONO-27). Mutation profiles were sequenced by a cancer gene-targeted NGS panel. Results: The tumor mutation burden(TMB) of the MSI-H CRC patients was significantly higher than those MSS CRC patients. Compared with the MSS CRC, MSI-H CRC involved more genes and pathways. Furthermore, we found the copy number variation (CNV) was different between the two groups. The copy number instability (CNI) score of MSI-H CRC patients was significantly lower than those MSS CRC patients. MSI-H CRC patients showed a higher frequency of TP53 gene CNV gain compared with MSS CRC (41% (7/17) in MSI-H CRC versus 13% (3/23) in MSS CRC). Conclusions: The mutational landscape are different between the MSI-H and MSS colorectal cancer. Compared with MSS colorectal cancer, the MSI-H colorectal cancer patients have higher tumor mutation burden(TMB) and lower copy number instability (CNI) score. Keywords: colorectal cancer, microsatellite instability, tumor mutation burden, copy number instability. Abbreviations CRC, colorectal cancer; TMB, tumor mutation burden; MSI-H, The microsatellite instability-high; MSS, microsatellite stable; CNV, copy number variation.