The lack of negative association between TE load and subgenome dominance in synthesized Brassica allotetraploids.

Polyploidization is important to the evolution of plants. Subgenome dominance is a distinct phenomenon associated with most allopolyploids. A gene on the dominant subgenome tends to express to higher RNA levels in all organs as compared to the expression of its syntenic paralogue (homoeolog). The mechanism that underlies the formation of subgenome dominance remains unknown, but there is evidence for the involvement of transposon/DNA methylation density differences nearby the genes of parents as being causal. The subgenome with lower density of transposon and methylation near genes is positively associated with subgenome dominance. Here, we generated eight generations of allotetraploid progenies from the merging of parental genomes Brassica rapa and Brassica oleracea. We found that transposon/methylation density differ near genes between the parental (rapa:oleracea) existed in the wide hybrid, persisted in the neotetraploids (the synthetic Brassica napus), but these neotetraploids expressed no expected subgenome dominance. This absence of B. rapa vs. B. oleracea subgenome dominance is particularly significant because, while there is no negative relationship between transposon/methylation level and subgenome dominance in the neotetraploids, the more ancient parental subgenomes for all Brassica did show differences in transposon/methylation densities near genes and did express, in the same samples of cells, biased gene expression diagnostic of subgenome dominance. We conclude that subgenome differences in methylated transposon near genes are not sufficient to initiate the biased gene expressions defining subgenome dominance. Our result was unexpected, and we suggest a "nuclear chimera" model to explain our data.

Screening and identification of female-specific DNA sequences in octaploid sturgeon using comparative genomics with high-throughput sequencing.

In this study, six candidate female-specific DNA sequences of octaploid Amur sturgeon (Acipenser schrenckii) were identified using comparative genomic approaches with high-throughput sequencing data. Their specificity was confirmed by traditional PCR. Two of these sex-specific sequences were also validated as female-specific in other eight sturgeon species and two hybrid sturgeons. The identified female-specific DNA fragments suggest that the family Acipenseridae has a ZZ/ZW sex-determining system. However, one of the two DNA sequences has been deleted in some sturgeons such as Sterlet sturgeon (Acipenser ruthenus), Beluga (Huso huso) and Kaluga (H. dauricus). The difference of sex-specific sequences among sturgeons indicates that there are different sex-specific regions among species of sturgeon. This study not only provided the sex-specific DNA sequences for management, conservation and studies of sex-determination mechanisms in sturgeons, but also confirmed the capability of the workflow to identify sex-specific DNA sequences in the polyploid species with complex genomes.

From asymmetrical to balanced genomic diversification during rediploidization: Subgenomic evolution in allotetraploid fish.

A persistent enigma is the rarity of polyploidy in animals, compared to its prevalence in plants. Although animal polyploids are thought to experience deleterious genomic chaos during initial polyploidization and subsequent rediploidization processes, this hypothesis has not been tested. We provide an improved reference-quality de novo genome for allotetraploid goldfish whose origin dates to ~15 million years ago. Comprehensive analyses identify changes in subgenomic evolution from asymmetrical oscillation in goldfish and common carp to diverse stabilization and balanced gene expression during continuous rediploidization. The homoeologs are coexpressed in most pathways, and their expression dominance shifts temporally during embryogenesis. Homoeolog expression correlates negatively with alternation of DNA methylation. The results show that allotetraploid cyprinids have a unique strategy for balancing subgenomic stabilization and diversification. Rediploidization process in these fishes provides intriguing insights into genome evolution and function in allopolyploid vertebrates.

Genetic Load and Potential Mutational Meltdown in Cancer Cell Populations.

Large genomes with elevated mutation rates are prone to accumulating deleterious mutations more rapidly than natural selection can purge (Muller's ratchet). As a consequence, it may lead to the extinction of small populations. Relative to most unicellular organisms, cancer cells, with large and nonrecombining genome and high mutation rate, could be particularly susceptible to such "mutational meltdown." However, the most common type of mutation in organismal evolution, namely, deleterious mutation, has received relatively little attention in the cancer biology literature. Here, by monitoring single-cell clones from HeLa cell lines, we characterize deleterious mutations that retard the rate of cell proliferation. The main mutation events are copy number variations (CNVs), which, estimated from fitness data, happen at a rate of 0.29 event per cell division on average. The mean fitness reduction, estimated reaching 18% per mutation, is very high. HeLa cell populations therefore have very substantial genetic load and, at this level, natural population would likely face mutational meltdown. We suspect that HeLa cell populations may avoid extinction only after the population size becomes large enough. Because CNVs are common in most cell lines and tumor tissues, the observations hint at cancer cells' vulnerability, which could be exploited by therapeutic strategies.

Ultrasensitive and high-efficiency screen of de novo low-frequency mutations by o2n-seq.

Detection of de novo, low-frequency mutations is essential for characterizing cancer genomes and heterogeneous cell populations. However, the screening capacity of current ultrasensitive NGS methods is inadequate owing to either low-efficiency read utilization or severe amplification bias. Here, we present o2n-seq, an ultrasensitive and high-efficiency NGS library preparation method for discovering de novo, low-frequency mutations. O2n-seq reduces the error rate of NGS to 10-5-10-8. The efficiency of its data usage is about 10-30 times higher than that of barcode-based strategies. For detecting mutations with allele frequency (AF) 1% in 4.6 Mb-sized genome, the sensitivity and specificity of o2n-seq reach to 99% and 98.64%, respectively. For mutations with AF around 0.07% in phix174, o2n-seq detects all the mutations with 100% specificity. Moreover, we successfully apply o2n-seq to screen de novo, low-frequency mutations in human tumours. O2n-seq will aid to characterize the landscape of somatic mutations in research and clinical settings.

novoBreak: local assembly for breakpoint detection in cancer genomes.

We present novoBreak, a genome-wide local assembly algorithm that discovers somatic and germline structural variation breakpoints in whole-genome sequencing data. novoBreak consistently outperformed existing algorithms on real cancer genome data and on synthetic tumors in the ICGC-TCGA DREAM 8.5 Somatic Mutation Calling Challenge primarily because it more effectively utilized reads spanning breakpoints. novoBreak also demonstrated great sensitivity in identifying short insertions and deletions.

Ultra-precise detection of mutations by droplet-based amplification of circularized DNA.

BACKGROUND: NGS (next generation sequencing) has been widely used in studies of biological processes, ranging from microbial evolution to cancer genomics. However, the error rate of NGS (0.1 % ~ 1 %) is still remaining a great challenge for comprehensively investigating the low frequency variations, and the current solution methods have suffered severe amplification bias or low efficiency. RESULTS: We creatively developed Droplet-CirSeq for relatively efficient, low-bias and ultra-sensitive identification of variations by combining millions of picoliter uniform-sized droplets with Cir-seq. Droplet-CirSeq is entitled with an incredibly low error rate of 3 ~ 5 X 10(-6). To systematically evaluate the performances of amplification uniformity and capability of mutation identification for Droplet-CirSeq, we took the mixtures of two E. coli strains as specific instances to simulate the circumstances of mutations with different frequencies. Compared with Cir-seq, the coefficient of variance of read depth for Droplet-CirSeq was 10 times less (p = 2.6 X 10(-3)), and the identified allele frequency presented more concentrated to the authentic frequency of mixtures (p = 4.8 X 10(-3)), illustrating a significant improvement of amplification bias and accuracy in allele frequency determination. Additionally, Droplet-CirSeq detected 2.5 times genuine SNPs (p < 0.001), achieved a 2.8 times lower false positive rate (p < 0.05) and a 1.5 times lower false negative rate (p < 0.001), in the case of a 3 pg DNA input. Intriguingly, the false positive sites predominantly represented in two types of base substitutions (G- > A, C- > T). Our findings indicated that 30 pg DNA input accommodated in 5 ~ 10 million droplets resulted in maximal detection of authentic mutations compared to 3 pg (p = 1.2 X 10(-8)) and 300 pg input (p = 2.2 X 10(-3)). CONCLUSIONS: We developed a method namely Droplet-CirSeq to significantly improve the amplification bias, which presents obvious superiority over the currently prevalent methods in exploitation of ultra-low frequency mutations. Droplet-CirSeq would be promisingly used in the identification of low frequency mutations initiated from extremely low input DNA, such as DNA of uncultured microorganisms, captured DNA of target region, circulation DNA of plasma et al, and its creative conception of rolling circle amplification in droplets would also be used in other low input DNA amplification fields.

Determination of genomic copy number alteration emphasizing a restriction site-based strategy of genome re-sequencing.

MOTIVATION: Copy number abbreviation (CNA) is one type of genomic aberration that is often induced by genome instability and is associated with diseases such as cancer. Determination of the genome-wide CNA profile is an important step in identifying the underlying mutation mechanisms. Genomic data based on next-generation sequencing technology are particularly suitable for determination of high-quality CNA profile. Now is an important time to reevaluate the use of sequencing techniques for CNA analysis, especially with the rapid growth of the different targeted genome and whole-genome sequencing strategies. RESULTS: In this study, we provide a comparison of resequencing strategies, with regard to their utility, applied to the same hepatocellular carcinoma sample for copy number determination. These strategies include whole-genome, exome and restriction site-associated DNA (RAD) sequencing. The last of these strategies is a targeted sequencing technique that involves cutting the genome with a restriction enzyme and isolating the targeted sequences. Our data demonstrate that RAD sequencing is an efficient and comprehensive strategy that allows the cost-effective determination of CNAs. Further investigation of RAD sequencing data led to the finding that a precise measurement of the allele frequency would be a helpful complement to the read depth for CNA analysis for two reasons. First, knowledge of the allele frequency helps to resolve refined calculations of allele-specific copy numbers, which, in turn, identify the functionally important CNAs that are under natural selection on the parental alleles. Second, this knowledge enables deconvolution of CNA patterns in complex genomic regions.

Identification of medium-sized genomic deletions with low coverage, mate-paired restricted tags.

BACKGROUND: Genomic deletions are known to be widespread in many species. Variant sequencing-based approaches for identifying deletions have been developed, but their powers to detect those deletions that affect medium-sized regions are limited when the sequencing coverage is low. RESULTS: We present a cost-effective method for identifying medium-sized deletions in genomic regions with low genomic coverage. Two mate-paired libraries were separately constructed from human cancerous tissue to generate paired short reads (ditags) from restriction fragments digested with a 4-base restriction enzyme. A total of 3 Gb of paired reads (1.0× genome size) was collected, and 175 deletions were inferred by identifying the ditags with disorder alignments to the reference genome sequence. Sanger sequencing results confirmed an overall detection accuracy of 95%. Good reproducibility was verified by the deletions that were detected by both libraries. CONCLUSIONS: We provide an approach to accurately identify medium-sized deletions in large genomes with low sequence coverage. It can be applied in studies of comparative genomics and in the identification of germline and somatic variants.

Rapid growth of a hepatocellular carcinoma and the driving mutations revealed by cell-population genetic analysis of whole-genome data.

We present the analysis of the evolution of tumors in a case of hepatocellular carcinoma. This case is particularly informative about cancer growth dynamics and the underlying driving mutations. We sampled nine different sections from three tumors and seven more sections from the adjacent nontumor tissues. Selected sections were subjected to exon as well as whole-genome sequencing. Putative somatic mutations were then individually validated across all 9 tumor and 7 nontumor sections. Among the mutations validated, 24 were amino acid changes; in addition, 22 large indels/copy number variants (>1 Mb) were detected. These somatic mutations define four evolutionary lineages among tumor cells. Separate evolution and expansion of these lineages were recent and rapid, each apparently having only one lineage-specific protein-coding mutation. Hence, by using a cell-population genetic definition, this approach identified three coding changes (CCNG1, P62, and an indel/fusion gene) as tumor driver mutations. These three mutations, affecting cell cycle control and apoptosis, are functionally distinct from mutations that accumulated earlier, many of which are involved in inflammation/immunity or cell anchoring. These distinct functions of mutations at different stages may reflect the genetic interactions underlying tumor growth.