Higher TP53BP2 expression is associated with HBsAg loss in peginterferon-α-treated patients with chronic hepatitis B.

BACKGROUND & AIMS: HBsAg loss is only observed in a small proportion of patients with chronic hepatitis B (CHB) who undergo interferon treatment. Investigating the host factors crucial for functional cure of CHB can aid in identifying individuals who would benefit from peginterferon-α (Peg-IFNα) therapy. METHODS: We conducted a genome-wide association study (GWAS) by enrolling 48 patients with CHB who achieved HBsAg loss after Peg-IFNα treatment and 47 patients who didn't. In the validation stage, we included 224 patients, of whom 90 had achieved HBsAg loss, to validate the identified significant single nucleotide polymorphisms. To verify the functional involvement of the candidate genes identified, we performed a series of in vitro and in vivo experiments. RESULTS: GWAS results indicated a significant association between the rs7519753 C allele and serum HBsAg loss in patients with CHB after Peg-IFNα treatment (p = 4.85 × 10-8, odds ratio = 14.47). This association was also observed in two independent validation cohorts. Expression quantitative trait locus analysis revealed higher hepatic TP53BP2 expression in individuals carrying the rs7519753 C allele (p = 2.90 × 10-6). RNA-sequencing of liver biopsies from patients with CHB after Peg-IFNα treatment revealed that hepatic TP53BP2 levels were significantly higher in the HBsAg loss group compared to the HBsAg persistence group (p = 0.035). In vitro and in vivo experiments demonstrated that loss of TP53BP2 decreased interferon-stimulated gene levels and the anti-HBV effect of IFN-α. Mechanistically, TP53BP2 was found to downregulate SOCS2, thereby facilitating JAK/STAT signaling. CONCLUSION: The rs7519753 C allele is associated with elevated hepatic TP53BP2 expression and an increased probability of serum HBsAg loss post-Peg-IFNα treatment in patients with CHB. TP53BP2 enhances the response of the hepatocyte to IFN-α by suppressing SOCS2 expression. IMPACT AND IMPLICATIONS: Chronic hepatitis B (CHB) remains a global public health issue. Although current antiviral therapies are more effective in halting disease progression, only a few patients achieve functional cure for hepatitis B with HBsAg loss, highlighting the urgent need for a cure for CHB. This study revealed that the rs7519753 C allele, which is associated with high expression of hepatic TP53BP2, significantly increases the likelihood of serum HBsAg loss in patients with CHB undergoing Peg-IFNα treatment. This finding not only provides a promising predictor for HBsAg loss but identifies a potential therapeutic target for Peg-IFNα treatment. We believe our results are of great interest to a wide range of stakeholders based on their potential clinical implications.

Noninvasive prenatal testing of α-thalassemia and ß-thalassemia through population-based parental haplotyping.

BACKGROUND: Noninvasive prenatal testing (NIPT) of recessive monogenic diseases depends heavily on knowing the correct parental haplotypes. However, the currently used family-based haplotyping method requires pedigrees, and molecular haplotyping is highly challenging due to its high cost, long turnaround time, and complexity. Here, we proposed a new two-step approach, population-based haplotyping-NIPT (PBH-NIPT), using α-thalassemia and ß-thalassemia as prototypes. METHODS: First, we deduced parental haplotypes with Beagle 4.0 with training on a large retrospective carrier screening dataset (4356 thalassemia carrier screening-positive cases). Second, we inferred fetal haplotypes using a parental haplotype-assisted hidden Markov model (HMM) and the Viterbi algorithm. RESULTS: With this approach, we enrolled 59 couples at risk of having a fetus with thalassemia and successfully inferred 94.1% (111/118) of fetal alleles. We confirmed these alleles by invasive prenatal diagnosis, with 99.1% (110/111) accuracy (95% CI, 95.1-100%). CONCLUSIONS: These results demonstrate that PBH-NIPT is a sensitive, fast, and inexpensive strategy for NIPT of thalassemia.

An efficient pipeline for ancient DNA mapping and recovery of endogenous ancient DNA from whole-genome sequencing data.

Ancient DNA research has developed rapidly over the past few decades due to improvements in PCR and next-generation sequencing (NGS) technologies, but challenges still exist. One major challenge in relation to ancient DNA research is to recover genuine endogenous ancient DNA sequences from raw sequencing data. This is often difficult due to degradation of ancient DNA and high levels of contamination, especially homologous contamination that has extremely similar genetic background with that of the real ancient DNA. In this study, we collected whole-genome sequencing (WGS) data from 6 ancient samples to compare different mapping algorithms. To further explore more effective methods to separate endogenous DNA from homologous contaminations, we attempted to recover reads based on ancient DNA specific characteristics of deamination, depurination, and DNA fragmentation with different parameters. We propose a quick and improved pipeline for separating endogenous ancient DNA while simultaneously decreasing homologous contaminations to very low proportions. Our goal in this research was to develop useful recommendations for ancient DNA mapping and for separation of endogenous DNA to facilitate future studies of ancient DNA.

Whole-genome sequencing of 175 Mongolians uncovers population-specific genetic architecture and gene flow throughout North and East Asia.

The genetic variation in Northern Asian populations is currently undersampled. To address this, we generated a new genetic variation reference panel by whole-genome sequencing of 175 ethnic Mongolians, representing six tribes. The cataloged variation in the panel shows strong population stratification among these tribes, which correlates with the diverse demographic histories in the region. Incorporating our results with the 1000 Genomes Project panel identifies derived alleles shared between Finns and Mongolians/Siberians, suggesting that substantial gene flow between northern Eurasian populations has occurred in the past. Furthermore, we highlight that North, East, and Southeast Asian populations are more aligned with each other than these groups are with South Asian and Oceanian populations.

Balanced Chromosomal Rearrangement Detection by Low-Pass Whole-Genome Sequencing.

Balanced chromosomal rearrangements (or balanced chromosome abnormalities, BCAs) are common chromosomal structural variants. Emerging studies have demonstrated the feasibility of using whole-genome sequencing (WGS) for detection of BCA-associated breakpoints, but the requirement for a priori knowledge of the rearranged regions from G-banded chromosome analysis limits its application. The protocols described here are based on low-pass WGS for detecting BCA events independent from chromosome analysis, and has been validated using genomic data from the 1000 Genomes Project. This approach adopts non-size-selected mate-pair library (3∼8 kb) with 2∼3 µg DNA as input, and requires only 30 million read-pairs (50 bp, equivalent to 1-fold base-coverage) for each sample. The complete procedure takes 13 days and the total cost is estimated to be less than $600 (USD) per sample. © 2018 by John Wiley & Sons, Inc.

Construction of the third-generation Zea mays haplotype map.

Background: Characterization of genetic variations in maize has been challenging, mainly due to deterioration of collinearity between individual genomes in the species. An international consortium of maize research groups combined resources to develop the maize haplotype version 3 (HapMap 3), built from whole-genome sequencing data from 1218 maize lines, covering predomestication and domesticated Zea mays varieties across the world. Results: A new computational pipeline was set up to process more than 12 trillion bp of sequencing data, and a set of population genetics filters was applied to identify more than 83 million variant sites. Conclusions: We identified polymorphisms in regions where collinearity is largely preserved in the maize species. However, the fact that the B73 genome used as the reference only represents a fraction of all haplotypes is still an important limiting factor.

Identification of balanced chromosomal rearrangements previously unknown among participants in the 1000 Genomes Project: implications for interpretation of structural variation in genomes and the future of clinical cytogenetics.

PURPOSE: Recent studies demonstrate that whole-genome sequencing enables detection of cryptic rearrangements in apparently balanced chromosomal rearrangements (also known as balanced chromosomal abnormalities, BCAs) previously identified by conventional cytogenetic methods. We aimed to assess our analytical tool for detecting BCAs in the 1000 Genomes Project without knowing which bands were affected. METHODS: The 1000 Genomes Project provides an unprecedented integrated map of structural variants in phenotypically normal subjects, but there is no information on potential inclusion of subjects with apparent BCAs akin to those traditionally detected in diagnostic cytogenetics laboratories. We applied our analytical tool to 1,166 genomes from the 1000 Genomes Project with sufficient physical coverage (8.25-fold). RESULTS: With this approach, we detected four reciprocal balanced translocations and four inversions, ranging in size from 57.9 kb to 13.3 Mb, all of which were confirmed by cytogenetic methods and polymerase chain reaction studies. One of these DNAs has a subtle translocation that is not readily identified by chromosome analysis because of the similarity of the banding patterns and size of exchanged segments, and another results in disruption of all transcripts of an OMIM gene. CONCLUSION: Our study demonstrates the extension of utilizing low-pass whole-genome sequencing for unbiased detection of BCAs including translocations and inversions previously unknown in the 1000 Genomes Project.

Population-wide sampling of retrotransposon insertion polymorphisms using deep sequencing and efficient detection.

Active retrotransposons play important roles during evolution and continue to shape our genomes today, especially in genetic polymorphisms underlying a diverse set of diseases. However, studies of human retrotransposon insertion polymorphisms (RIPs) based on whole-genome deep sequencing at the population level have not been sufficiently undertaken, despite the obvious need for a thorough characterization of RIPs in the general population. Herein, we present a novel and efficient computational tool called Specific Insertions Detector (SID) for the detection of non-reference RIPs. We demonstrate that SID is suitable for high-depth whole-genome sequencing data using paired-end reads obtained from simulated and real datasets. We construct a comprehensive RIP database using a large population of 90 Han Chinese individuals with a mean ×68 depth per individual. In total, we identify 9342 recent RIPs, and 8433 of these RIPs are novel compared with dbRIP, including 5826 Alu, 2169 long interspersed nuclear element 1 (L1), 383 SVA, and 55 long terminal repeats. Among the 9342 RIPs, 4828 were located in gene regions and 5 were located in protein-coding regions. We demonstrate that RIPs can, in principle, be an informative resource to perform population evolution and phylogenetic analyses. Taking the demographic effects into account, we identify a weak negative selection on SVA and L1 but an approximately neutral selection for Alu elements based on the frequency spectrum of RIPs. SID is a powerful open-source program for the detection of non-reference RIPs. We built a non-reference RIP dataset that greatly enhanced the diversity of RIPs detected in the general population, and it should be invaluable to researchers interested in many aspects of human evolution, genetics, and disease. As a proof of concept, we demonstrate that the RIPs can be used as biomarkers in a similar way as single nucleotide polymorphisms.

Deep whole-genome sequencing of 90 Han Chinese genomes.

Next-generation sequencing provides a high-resolution insight into human genetic information. However, the focus of previous studies has primarily been on low-coverage data due to the high cost of sequencing. Although the 1000 Genomes Project and the Haplotype Reference Consortium have both provided powerful reference panels for imputation, low-frequency and novel variants remain difficult to discover and call with accuracy on the basis of low-coverage data. Deep sequencing provides an optimal solution for the problem of these low-frequency and novel variants. Although whole-exome sequencing is also a viable choice for exome regions, it cannot account for noncoding regions, sometimes resulting in the absence of important, causal variants. For Han Chinese populations, the majority of variants have been discovered based upon low-coverage data from the 1000 Genomes Project. However, high-coverage, whole-genome sequencing data are limited for any population, and a large amount of low-frequency, population-specific variants remain uncharacterized. We have performed whole-genome sequencing at a high depth (∼×80) of 90 unrelated individuals of Chinese ancestry, collected from the 1000 Genomes Project samples, including 45 Northern Han Chinese and 45 Southern Han Chinese samples. Eighty-three of these 90 have been sequenced by the 1000 Genomes Project. We have identified 12 568 804 single nucleotide polymorphisms, 2 074 210 short InDels, and 26 142 structural variations from these 90 samples. Compared to the Han Chinese data from the 1000 Genomes Project, we have found 7 000 629 novel variants with low frequency (defined as minor allele frequency < 5%), including 5 813 503 single nucleotide polymorphisms, 1 169 199 InDels, and 17 927 structural variants. Using deep sequencing data, we have built a greatly expanded spectrum of genetic variation for the Han Chinese genome. Compared to the 1000 Genomes Project, these Han Chinese deep sequencing data enhance the characterization of a large number of low-frequency, novel variants. This will be a valuable resource for promoting Chinese genetics research and medical development. Additionally, it will provide a valuable supplement to the 1000 Genomes Project, as well as to other human genome projects.

Sequencing and de novo assembly of 150 genomes from Denmark as a population reference.

Hundreds of thousands of human genomes are now being sequenced to characterize genetic variation and use this information to augment association mapping studies of complex disorders and other phenotypic traits. Genetic variation is identified mainly by mapping short reads to the reference genome or by performing local assembly. However, these approaches are biased against discovery of structural variants and variation in the more complex parts of the genome. Hence, large-scale de novo assembly is needed. Here we show that it is possible to construct excellent de novo assemblies from high-coverage sequencing with mate-pair libraries extending up to 20 kilobases. We report de novo assemblies of 150 individuals (50 trios) from the GenomeDenmark project. The quality of these assemblies is similar to those obtained using the more expensive long-read technology. We use the assemblies to identify a rich set of structural variants including many novel insertions and demonstrate how this variant catalogue enables further deciphering of known association mapping signals. We leverage the assemblies to provide 100 completely resolved major histocompatibility complex haplotypes and to resolve major parts of the Y chromosome. Our study provides a regional reference genome that we expect will improve the power of future association mapping studies and hence pave the way for precision medicine initiatives, which now are being launched in many countries including Denmark.

A Genetic Mechanism for Convergent Skin Lightening during Recent Human Evolution.

Skin lightening among Eurasians is thought to have been a convergence occurring independently in Europe and East Asia as an adaptation to high latitude environments. Among Europeans, several genes responsible for such lightening have been found, but the information available for East Asians is much more limited. Here, a genome-wide comparison between dark-skinned Africans and Austro-Asiatic speaking aborigines and light-skinned northern Han Chinese identified the pigmentation gene OCA2, showing unusually deep allelic divergence between these groups. An amino acid substitution (His615Arg) of OCA2 prevalent in most East Asian populations-but absent in Africans and Europeans-was significantly associated with skin lightening among northern Han Chinese. Further transgenic and targeted gene modification analyses of zebrafish and mouse both exhibited the phenotypic effect of the OCA2 variant manifesting decreased melanin production. These results indicate that OCA2 plays an important role in the convergent skin lightening of East Asians during recent human evolution.

Association Analysis of Genetic Variants with Type 2 Diabetes in a Mongolian Population in China.

The large scale genome wide association studies (GWAS) have identified approximately 80 single nucleotide polymorphisms (SNPs) conferring susceptibility to type 2 diabetes (T2D). However, most of these loci have not been replicated in diverse populations and much genetic heterogeneity has been observed across ethnic groups. We tested 28 SNPs previously found to be associated with T2D by GWAS in a Mongolian sample of Northern China (497 diagnosed with T2D and 469 controls) for association with T2D and diabetes related quantitative traits. We replicated T2D association of 11 SNPs, namely, rs7578326 (IRS1), rs1531343 (HMGA2), rs8042680 (PRC1), rs7578597 (THADA), rs1333051 (CDKN2), rs6723108 (TMEM163), rs163182 and rs2237897 (KCNQ1), rs1387153 (MTNR1B), rs243021 (BCL11A), and rs10229583 (PAX4) in our sample. Further, we showed that risk allele of the strongest T2D associated SNP in our sample, rs757832 (IRS1), is associated with increased level of TG. We observed substantial difference of T2D risk allele frequency between the Mongolian sample and the 1000G Caucasian sample for a few SNPs, including rs6723108 (TMEM163) whose risk allele reaches near fixation in the Mongolian sample. Further study of genetic architecture of these variants in susceptibility of T2D is needed to understand the role of these variants in heterogeneous populations.

The genome of a Mongolian individual reveals the genetic imprints of Mongolians on modern human populations.

Mongolians have played a significant role in modern human evolution, especially after the rise of Genghis Khan (1162[?]-1227). Although the social cultural impacts of Genghis Khan and the Mongolian population have been well documented, explorations of their genome structure and genetic imprints on other human populations have been lacking. We here present the genome of a Mongolian male individual. The genome was de novo assembled using a total of 130.8-fold genomic data produced from massively parallel whole-genome sequencing. We identified high-confidence variation sets, including 3.7 million single nucleotide polymorphisms (SNPs) and 756,234 short insertions and deletions. Functional SNP analysis predicted that the individual has a pathogenic risk for carnitine deficiency. We located the patrilineal inheritance of the Mongolian genome to the lineage D3a through Y haplogroup analysis and inferred that the individual has a common patrilineal ancestor with Tibeto-Burman populations and is likely to be the progeny of the earliest settlers in East Asia. We finally investigated the genetic imprints of Mongolians on other human populations using different approaches. We found varying degrees of gene flows between Mongolians and populations living in Europe, South/Central Asia, and the Indian subcontinent. The analyses demonstrate that the genetic impacts of Mongolians likely resulted from the expansion of the Mongolian Empire in the 13th century. The genome will be of great help in further explorations of modern human evolution and genetic causes of diseases/traits specific to Mongolians.

Exome capture from saliva produces high quality genomic and metagenomic data.

BACKGROUND: Targeted capture of genomic regions reduces sequencing cost while generating higher coverage by allowing biomedical researchers to focus on specific loci of interest, such as exons. Targeted capture also has the potential to facilitate the generation of genomic data from DNA collected via saliva or buccal cells. DNA samples derived from these cell types tend to have a lower human DNA yield, may be degraded from age and/or have contamination from bacteria or other ambient oral microbiota. However, thousands of samples have been previously collected from these cell types, and saliva collection has the advantage that it is a non-invasive and appropriate for a wide variety of research. RESULTS: We demonstrate successful enrichment and sequencing of 15 South African KhoeSan exomes and 2 full genomes with samples initially derived from saliva. The expanded exome dataset enables us to characterize genetic diversity free from ascertainment bias for multiple KhoeSan populations, including new exome data from six HGDP Namibian San, revealing substantial population structure across the Kalahari Desert region. Additionally, we discover and independently verify thirty-one previously unknown KIR alleles using methods we developed to accurately map and call the highly polymorphic HLA and KIR loci from exome capture data. Finally, we show that exome capture of saliva-derived DNA yields sufficient non-human sequences to characterize oral microbial communities, including detection of bacteria linked to oral disease (e.g. Prevotella melaninogenica). For comparison, two samples were sequenced using standard full genome library preparation without exome capture and we found no systematic bias of metagenomic information between exome-captured and non-captured data. CONCLUSIONS: DNA from human saliva samples, collected and extracted using standard procedures, can be used to successfully sequence high quality human exomes, and metagenomic data can be derived from non-human reads. We find that individuals from the Kalahari carry a higher oral pathogenic microbial load than samples surveyed in the Human Microbiome Project. Additionally, rare variants present in the exomes suggest strong population structure across different KhoeSan populations.

Identification of candidate genes for drought tolerance by whole-genome resequencing in maize.

BACKGROUND: Drought stress is one of the major limiting factors for maize production. With the availability of maize B73 reference genome and whole-genome resequencing of 15 maize inbreds, common variants (CV) and clustering analyses were applied to identify non-synonymous SNPs (nsSNPs) and corresponding candidate genes for drought tolerance. RESULTS: A total of 524 nsSNPs that were associated with 271 candidate genes involved in plant hormone regulation, carbohydrate and sugar metabolism, signaling molecules regulation, redox reaction and acclimation of photosynthesis to environment were detected by CV and cluster analyses. Most of the nsSNPs identified were clustered in bin 1.07 region that harbored six previously reported QTL with relatively high phenotypic variation explained for drought tolerance. Genes Ontology (GO) analysis of candidate genes revealed that there were 35 GO terms related to biotic stimulus and membrane-bounded organelle, showing significant differences between the candidate genes and the reference B73 background. Changes of expression level in these candidate genes for drought tolerance were detected using RNA sequencing for fertilized ovary, basal leaf meristem tissue and roots collected under drought stressed and well-watered conditions. The results indicated that 70% of candidate genes showed significantly expression changes under two water treatments and our strategies for mining candidate genes are feasible and relatively efficient. CONCLUSIONS: Our results successfully revealed candidate nsSNPs and associated genes for drought tolerance by comparative sequence analysis of 16 maize inbred lines. Both methods we applied were proved to be efficient for identifying candidate genes for complex traits through the next-generation sequencing technologies (NGS). These selected genes will not only facilitate understanding of genetic basis of drought stress response, but also accelerate genetic improvement through marker-assisted selection in maize.

Dissecting yield-associated loci in super hybrid rice by resequencing recombinant inbred lines and improving parental genome sequences.

The growing world population and shrinkage of arable land demand yield improvement of rice, one of the most important staple crops. To elucidate the genetic basis of yield and uncover its associated loci in rice, we resequenced the core recombinant inbred lines of Liang-You-Pei-Jiu, the widely cultivated super hybrid rice, and constructed a high-resolution linkage map. We detected 43 yield-associated quantitative trait loci, of which 20 are unique. Based on the high-density physical map, the genome sequences of paternal variety 93-11 and maternal cultivar PA64s of Liang-You-Pei-Jiu were significantly improved. The large recombinant inbred line population combined with plentiful high-quality single nucleotide polymorphisms and insertions/deletions between parental genomes allowed us to fine-map two quantitative trait loci, qSN8 and qSPB1, and to identify days to heading8 and lax panicle1 as candidate genes, respectively. The quantitative trait locus qSN8 was further confirmed to be days to heading8 by a complementation test. Our study provided an ideal platform for molecular breeding by targeting and dissecting yield-associated loci in rice.

Molecular footprints of domestication and improvement in soybean revealed by whole genome re-sequencing.

BACKGROUND: Artificial selection played an important role in the origin of modern Glycine max cultivars from the wild soybean Glycine soja. To elucidate the consequences of artificial selection accompanying the domestication and modern improvement of soybean, 25 new and 30 published whole-genome re-sequencing accessions, which represent wild, domesticated landrace, and Chinese elite soybean populations were analyzed. RESULTS: A total of 5,102,244 single nucleotide polymorphisms (SNPs) and 707,969 insertion/deletions were identified. Among the SNPs detected, 25.5% were not described previously. We found that artificial selection during domestication led to more pronounced reduction in the genetic diversity of soybean than the switch from landraces to elite cultivars. Only a small proportion (2.99%) of the whole genomic regions appear to be affected by artificial selection for preferred agricultural traits. The selection regions were not distributed randomly or uniformly throughout the genome. Instead, clusters of selection hotspots in certain genomic regions were observed. Moreover, a set of candidate genes (4.38% of the total annotated genes) significantly affected by selection underlying soybean domestication and genetic improvement were identified. CONCLUSIONS: Given the uniqueness of the soybean germplasm sequenced, this study drew a clear picture of human-mediated evolution of the soybean genomes. The genomic resources and information provided by this study would also facilitate the discovery of genes/loci underlying agronomically important traits.

Whole-genome sequences of DA and F344 rats with different susceptibilities to arthritis, autoimmunity, inflammation and cancer.

DA (D-blood group of Palm and Agouti, also known as Dark Agouti) and F344 (Fischer) are two inbred rat strains with differences in several phenotypes, including susceptibility to autoimmune disease models and inflammatory responses. While these strains have been extensively studied, little information is available about the DA and F344 genomes, as only the Brown Norway (BN) and spontaneously hypertensive rat strains have been sequenced to date. Here we report the sequencing of the DA and F344 genomes using next-generation Illumina paired-end read technology and the first de novo assembly of a rat genome. DA and F344 were sequenced with an average depth of 32-fold, covered 98.9% of the BN reference genome, and included 97.97% of known rat ESTs. New sequences could be assigned to 59 million positions with previously unknown data in the BN reference genome. Differences between DA, F344, and BN included 19 million positions in novel scaffolds, 4.09 million single nucleotide polymorphisms (SNPs) (including 1.37 million new SNPs), 458,224 short insertions and deletions, and 58,174 structural variants. Genetic differences between DA, F344, and BN, including high-impact SNPs and short insertions and deletions affecting >2500 genes, are likely to account for most of the phenotypic variation between these strains. The new DA and F344 genome sequencing data should facilitate gene discovery efforts in rat models of human disease.