Ruolan Qiu

Noninvasive Whole-Genome Sequencing of a Human Fetus

Sequencing of cell-free fetal-derived DNA from maternal plasma provides a noninvasive way to predict the fetal genome sequence. Not Your Mother’s Genome There are more than 3000 single-gene (Mendelian) disorders that are individually rare but collectively affect ~1% of births. Currently, only a few specific disorders are screened for during pregnancy, and definitive diagnosis requires invasive procedures such as amniocentesis. An ideal prenatal genetic diagnostic would noninvasively screen for all Mendelian disorders early in pregnancy. Exploiting the observation that ~10% of DNA floating freely in a pregnant woman’s plasma originates from the fetus she carries, several groups have developed DNA sequencing–based tests for conditions such as trisomy 21, the genetic cause of Down syndrome. Although these tests may readily detect gross abnormalities such as an extra copy of an entire chromosome, the noninvasive determination of a complete fetal genome sequence has remained out of reach. Here, Kitzman et al. reconstruct the whole-genome sequence of a human fetus using samples obtained noninvasively during the second trimester, including DNA from the pregnant mother, DNA from the father, and “cell-free” DNA from the pregnant mother’s plasma (a mixture of the maternal and fetal genomes). A big challenge for the authors was to be able to predict which genetic variants were passed from mother to fetus, because the overwhelming majority of DNA in the pregnant mother’s plasma derives from her genome rather than that of the fetus. To overcome this problem, the authors applied a recently developed technique to resolve the mother’s “haplotypes”—groups of genetic variants residing on the same chromosomes—and then used these groups to accurately predict inheritance. Another challenge was the identification of new mutations in the genome of the fetus. The authors demonstrate that, in principle, such mutations can be sensitively detected and triaged for validation. Although these methods must be refined and their costs driven down, this study hints that comprehensive, noninvasive prenatal screening for Mendelian disorders may be clinically feasible in the near future. Analysis of cell-free fetal DNA in maternal plasma holds promise for the development of noninvasive prenatal genetic diagnostics. Previous studies have been restricted to detection of fetal trisomies, to specific paternally inherited mutations, or to genotyping common polymorphisms using material obtained invasively, for example, through chorionic villus sampling. Here, we combine genome sequencing of two parents, genome-wide maternal haplotyping, and deep sequencing of maternal plasma DNA to noninvasively determine the genome sequence of a human fetus at 18.5 weeks of gestation. Inheritance was predicted at 2.8 × 106 parental heterozygous sites with 98.1% accuracy. Furthermore, 39 of 44 de novo point mutations in the fetal genome were detected, albeit with limited specificity. Subsampling these data and analyzing a second family trio by the same approach indicate that parental haplotype blocks of ~300 kilo–base pairs combined with shallow sequencing of maternal plasma DNA is sufficient to substantially determine the inherited complement of a fetal genome. However, ultradeep sequencing of maternal plasma DNA is necessary for the practical detection of fetal de novo mutations genome-wide. Although technical and analytical challenges remain, we anticipate that noninvasive analysis of inherited variation and de novo mutations in fetal genomes will facilitate prenatal diagnosis of both recessive and dominant Mendelian disorders.

Haplotype-resolved genome sequencing of a Gujarati Indian individual

Haplotype information is essential to the complete description and interpretation of genomes, genetic diversity and genetic ancestry. Although individual human genome sequencing is increasingly routine, nearly all such genomes are unresolved with respect to haplotype. Here we combine the throughput of massively parallel sequencing with the contiguity information provided by large-insert cloning to experimentally determine the haplotype-resolved genome of a South Asian individual. A single fosmid library was split into a modest number of pools, each providing ∼3% physical coverage of the diploid genome. Sequencing of each pool yielded reads overwhelmingly derived from only one homologous chromosome at any given location. These data were combined with whole-genome shotgun sequence to directly phase 94% of ascertained heterozygous single nucleotide polymorphisms (SNPs) into long haplotype blocks (N50 of 386 kilobases (kbp)). This method also facilitates the analysis of structural variation, for example, to anchor novel insertions to specific locations and haplotypes.

Chromosome-scale scaffolding of de novo genome assemblies based on chromatin interactions

Genomes assembled de novo from short reads are highly fragmented relative to the finished chromosomes of Homo sapiens and key model organisms generated by the Human Genome Project. To address this problem, we need scalable, cost-effective methods to obtain assemblies with chromosome-scale contiguity. Here we show that genome-wide chromatin interaction data sets, such as those generated by Hi-C, are a rich source of long-range information for assigning, ordering and orienting genomic sequences to chromosomes, including across centromeres. To exploit this finding, we developed an algorithm that uses Hi-C data for ultra-long-range scaffolding of de novo genome assemblies. We demonstrate the approach by combining shotgun fragment and short jump mate-pair sequences with Hi-C data to generate chromosome-scale de novo assemblies of the human, mouse and Drosophila genomes, achieving—for the human genome—98% accuracy in assigning scaffolds to chromosome groups and 99% accuracy in ordering and orienting scaffolds within chromosome groups. Hi-C data can also be used to validate chromosomal translocations in cancer genomes.

The haplotype-resolved genome and epigenome of the aneuploid HeLa cancer cell line

The HeLa cell line was established in 1951 from cervical cancer cells taken from a patient, Henrietta Lacks. This was the first successful attempt to immortalize human-derived cells in vitro. The robust growth and unrestricted distribution of HeLa cells resulted in its broad adoption—both intentionally and through widespread cross-contamination—and for the past 60 years it has served a role analogous to that of a model organism. The cumulative impact of the HeLa cell line on research is demonstrated by its occurrence in more than 74,000 PubMed abstracts (approximately 0.3%). The genomic architecture of HeLa remains largely unexplored beyond its karyotype, partly because like many cancers, its extensive aneuploidy renders such analyses challenging. We carried out haplotype-resolved whole-genome sequencing of the HeLa CCL-2 strain, examined point- and indel-mutation variations, mapped copy-number variations and loss of heterozygosity regions, and phased variants across full chromosome arms. We also investigated variation and copy-number profiles for HeLa S3 and eight additional strains. We find that HeLa is relatively stable in terms of point variation, with few new mutations accumulating after early passaging. Haplotype resolution facilitated reconstruction of an amplified, highly rearranged region of chromosome 8q24.21 at which integration of the human papilloma virus type 18 (HPV-18) genome occurred and that is likely to be the event that initiated tumorigenesis. We combined these maps with RNA-seq and ENCODE Project data sets to phase the HeLa epigenome. This revealed strong, haplotype-specific activation of the proto-oncogene MYC by the integrated HPV-18 genome approximately 500 kilobases upstream, and enabled global analyses of the relationship between gene dosage and expression. These data provide an extensively phased, high-quality reference genome for past and future experiments relying on HeLa, and demonstrate the value of haplotype resolution for characterizing cancer genomes and epigenomes.

Massively multiplex single-cell Hi-C

We present single-cell combinatorial indexed Hi-C (sciHi-C), a method that applies combinatorial cellular indexing to chromosome conformation capture. In this proof of concept, we generate and sequence six sciHi-C libraries comprising a total of 10,696 single cells. We use sciHi-C data to separate cells by karyotypic and cell-cycle state differences and identify cell-to-cell heterogeneity in mammalian chromosomal conformation. Our results demonstrate that combinatorial indexing is a generalizable strategy for single-cell genomics.

High-throughput determination of RNA structure by proximity ligation

We present an unbiased method to globally resolve RNA structures through pairwise contact measurements between interacting regions. RNA proximity ligation (RPL) uses proximity ligation of native RNA followed by deep sequencing to yield chimeric reads with ligation junctions in the vicinity of structurally proximate bases. We apply RPL in both bakers yeast (Saccharomyces cerevisiae) and human cells and generate contact probability maps for ribosomal and other abundant RNAs, including yeast snoRNAs, the RNA subunit of the signal recognition particle and the yeast U2 spliceosomal RNA homolog. RPL measurements correlate with established secondary structures for these RNA molecules, including stem-loop structures and long-range pseudoknots. We anticipate that RPL will complement the current repertoire of computational and experimental approaches in enabling the high-throughput determination of secondary and tertiary RNA structures.

Mapping 3D genome architecture through in situ DNase Hi-C

With the advent of massively parallel sequencing, considerable work has gone into adapting chromosome conformation capture (3C) techniques to study chromosomal architecture at a genome-wide scale. We recently demonstrated that the inactive murine X chromosome adopts a bipartite structure using a novel 3C protocol, termed in situ DNase Hi-C. Like traditional Hi-C protocols, in situ DNase Hi-C requires that chromatin be chemically cross-linked, digested, end-repaired, and proximity-ligated with a biotinylated bridge adaptor. The resulting ligation products are optionally sheared, affinity-purified via streptavidin bead immobilization, and subjected to traditional next-generation library preparation for Illumina paired-end sequencing. Importantly, in situ DNase Hi-C obviates the dependence on a restriction enzyme to digest chromatin, instead relying on the endonuclease DNase I. Libraries generated by in situ DNase Hi-C have a higher effective resolution than traditional Hi-C libraries, which makes them valuable in cases in which high sequencing depth is allowed for, or when hybrid capture technologies are expected to be used. The protocol described here, which involves ∼4 d of bench work, is optimized for the study of mammalian cells, but it can be broadly applicable to any cell or tissue of interest, given experimental parameter optimization.

Non-invasive fetal genome sequencing: Opportunities and challenges†

We recently predicted the whole genome sequence of a human fetus using samples obtained non-invasively from the pregnant mother and the father. [Kitzman et al., 2012] This advance raises the possibility that it may soon be possible to perform genome-wide prenatal genetic testing without an invasive procedure early in pregnancy. Such a test would substantially broaden the scope of fetal genetic results that could be available prenatally. Non-invasive fetal genome sequencing (NIFGS) does not inherently raise new ethical issues, or those that cannot be addressed within the existing framework of medical bioethics. Indeed, many of the same issues have been raised by the introduction of other prenatal testing / screening technologies, now in wide use, and again more recently by the introduction of whole genome sequencing for clinical diagnosis. [Sayres et al., 2011, Schmitz et al., 2009, Ravitsky, 2009, Benn et al., 2009, Tabor et al., 2011, Berg et al., 2011] However, the ethical issues are, somewhat, magnified by the possibility of NIFGS and compounded by controversies surrounding elective pregnancy termination, rights of individuals with disabilities, and eugenics. Accordingly, the prospect of successful NIFGS, even on a research basis, is likely to generate considerable controversy and debate about the acceptability of developing such technologies, much less if and how they should be used. We view this response as very positive because it provides all stakeholders and the broader public in general with the opportunity to carefully consider and deliberate these issues in what we would hope is a thoughtful and balanced way. As NIFGS becomes technically tractable and increasingly cost-effective, and as an acceptable false positive/false negative profile is achieved, one population for which it might be of great benefit may be pregnant women who are currently offered invasive prenatal diagnostic testing. Such women are typically at risk for genetic conditions based on screening results or family history, and NIFGS would likely reduce if not eliminate adverse outcomes from invasive testing for most of these women. The expanded use of NIFGS would present several advantages and challenges. Broader use of NIFGS might lead to the greater detection of Mendelian disorders in families who would not otherwise have been offered prenatal testing, as well as families who might have refused invasive testing because of risks to the pregnancy and fetus. NIFGS could augment or even replace current approaches to neonatal screening as most such disorders are autosomal or X-linked recessive (e.g., hypothyroidism and congenital hearing loss are only sometimes Mendelian). Prenatal identification of disorders now found in neonatal screening would afford for earlier parental education, diminished false positives and the accompanying costs of retesting and parental anxiety and earlier therapeutic interventions. Earlier detection of such disorders would also foster improved prenatal care, pregnancy and delivery management and/or postnatal intervention. For example, 90% of genetic variants in SCNA1 that cause seizure disorders are de novo, and identification by NIFGS could allow for diagnosis before the onset of seizures and consideration of appropriate precautions and/or pharmacological treatment. [Marini et al., 20011] Similarly, 50% of mutations causing Multiple Endocrine Neoplasia 2B are spontaneous, and earlier identification of these mutations could prompt prophylactic thyroidectomy and improve outcomes. [Carlson et al., 1994] The availability of NIFGS could increase the utilization of prenatal testing, and in turn increase rates of elective termination, both for disorders for which testing is currently available and for the wide arrange of disorders and traits for which testing would be newly available. [Tischler et al., 2011] On the other hand, NIFGS might also make pregnancy termination safer, less costly, and less traumatic as it could be performed early in gestation. Broad use of NIFGS might result in increased societal pressure for pregnant women to undergo screening and terminate any fetus suspected to have a Mendelian condition. This could reverse important and continuing social progress towards civil rights and social support for people and families with disabilities. In addition, this societal pressure might threaten parental autonomy over reproductive decision-making. Broader use of NIFGS might also create or magnify social stigmas or inequities. NIFGS would likely remain expensive and may not be reimbursable by insurance in the short-term. This might exaggerate disparities between people who can easily afford access and those who cannot. If access is limited to those who can afford it, it is possible that a disproportionate number of lower income families could suffer from the higher rates of morbidity and mortality of invasive testing. In the extreme scenario, children with Mendelian conditions would be disproportionately born to lower income families that could not afford NIFGS. Such a disparity would likely further stigmatize many of these conditions and exaggerate existing disparities in access to healthcare and benefits for these populations. Another key issue raised by NIFGS is that it represents a substantially more comprehensive test for Mendelian disorders with a known cause, and will identify variants that are beyond the scope of conventional prenatal screening and diagnosis. Specifically, variants will be identified that indicate increased risk for developing adult onset conditions. This is not unique to NIFGS: in fact this is an ongoing challenge in pediatric clinical genetic testing. [Wilfond et al., 2009] Such information may be irrelevant or inappropriate to return for the benefit of the fetus/future child, but may have direct implications for the health of the parent, and therefore provide indirect benefit to any current or future children. However, if NIFGS is more broadly implemented, the scope of the results identified and the number of individuals affected may increase substantially. This will further overwhelm the existing infrastructure for providing genetic counseling. As with other applications of whole-genome sequencing, NIFGS will identify variants of ambiguous clinical utility in genes known to be associated with both pediatric and adult complex disease. For example, Kitzman et al. found a de novo novel missense variant in ACMSD, a gene in which common variants have been associated with Parkinson disease by genome-wide association. [Klitzman et al., 2012, International Parkinson Disease Genomics Consortium et al., 2011] This variant causes substitution of a highly conserved amino acid residue, but in the absence of compelling evidence of its role in Parkinson disease or other conditions, its detection is of limited clinical value. While this is no different than the challenge of interpreting WGS information in general, pregnancy might be a particularly vulnerable time in which to receive this information and parents might feel compelled to give more credence to the information than it warrants. There are several other important issues that require consideration. Will the non-invasive nature of this test, combined with the enhanced detection of Mendelian disorders, lead to a substantial increase in the number of women who consider prenatal diagnosis? How will the medical community meet the challenge of providing genetic counseling to address the complex nature of the information that may be identified? These concerns raise the possibility that some women may not be able to provide adequate informed consent, or may proceed with actions such as terminations without complete understanding of the test results or the prognosis for various rare Mendelian disorders. If NIFGS allows the creation of a record of a child’s whole genome prior to its birth, what should happen to that data? Should it be stored as part of the child’s medical record, with the possibility for future updating, analysis and mining for medically relevant information? Or should it be destroyed? Who should make this decision and have control over the data? As with many new technologies, NIFGS will be accompanied by many ethical and social challenges. We think that it is imperative that these questions and issues be discussed and addressed by a diverse group of stakeholders, as well as through collection of empirical data on stakeholder perspectives and concerns. Much can be learned from the history of the implementation of other prenatal testing approaches, such as amniocentesis and CVS, as well as the ongoing debates about pediatric genetic testing and return of results from whole genome sequencing. [Rapp, 2000]

High-resolution comparative analysis of great ape genomes

A spotlight on great ape genomes Most nonhuman primate genomes generated to date have been “humanized” owing to their many gaps and the reliance on guidance by the reference human genome. To remove this humanizing effect, Kronenberg et al. generated and assembled long-read genomes of a chimpanzee, an orangutan, and two humans and compared them with a previously generated gorilla genome. This analysis recognized genomic structural variation specific to humans and particular ape lineages. Comparisons between human and chimpanzee cerebral organoids showed down-regulation of the expression of specific genes in humans, relative to chimpanzees, related to noncoding variation identified in this analysis. Science, this issue p. eaar6343 Analysis of long-read great ape and human genomes identifies human-specific changes affecting brain gene expression. INTRODUCTION Understanding the genetic differences that make us human is a long-standing endeavor that requires the comprehensive discovery and comparison of all forms of genetic variation within great ape lineages. RATIONALE The varied quality and completeness of ape genomes have limited comparative genetic analyses. To eliminate this contiguity and quality disparity, we generated human and nonhuman ape genome assemblies without the guidance of the human reference genome. These new genome assemblies enable both coarse and fine-scale comparative genomic studies. RESULTS We sequenced and assembled two human, one chimpanzee, and one orangutan genome using high-coverage (>65x) single-molecule, real-time (SMRT) long-read sequencing technology. We also sequenced more than 500,000 full-length complementary DNA samples from induced pluripotent stem cells to construct de novo gene models, increasing our knowledge of transcript diversity in each ape lineage. The new nonhuman ape genome assemblies improve gene annotation and genomic contiguity (by 30- to 500-fold), resulting in the identification of larger synteny blocks (by 22- to 74-fold) when compared to earlier assemblies. Including the latest gorilla genome, we now estimate that 83% of the ape genomes can be compared in a multiple sequence alignment. We observe a modest increase in single-nucleotide variant divergence compared to previous genome analyses and estimate that 36% of human autosomal DNA is subject to incomplete lineage sorting. We fully resolve most common repeat differences, including full-length retrotransposons such as the African ape-specific endogenous retroviral element PtERV1. We show that the spread of this element independently in the gorilla and chimpanzee lineage likely resulted from a founder element that failed to segregate to the human lineage because of incomplete lineage sorting. The improved sequence contiguity allowed a more systematic discovery of structural variation (>50 base pairs in length) (see the figure). We detected 614,186 ape deletions, insertions, and inversions, assigning each to specific ape lineages. Unbiased genome scaffolding (optical maps, bacterial artificial chromosome sequencing, and fluorescence in situ hybridization) led to the discovery of large, unknown complex inversions in gene-rich regions. Of the 17,789 fixed human-specific insertions and deletions, we focus on those of potential functional effect. We identify 90 that are predicted to disrupt genes and an additional 643 that likely affect regulatory regions, more than doubling the number of human-specific deletions that remove regulatory sequence in the human lineage. We investigate the association of structural variation with changes in human-chimpanzee brain gene expression using cerebral organoids as a proxy for expression differences. Genes associated with fixed structural variants (SVs) show a pattern of down-regulation in human radial glial neural progenitors, whereas human-specific duplications are associated with up-regulated genes in human radial glial and excitatory neurons (see the figure). CONCLUSION The improved ape genome assemblies provide the most comprehensive view to date of intermediate-size structural variation and highlight several dozen genes associated with structural variation and brain-expression differences between humans and chimpanzees. These new references will provide a stepping stone for the completion of great ape genomes at a quality commensurate with the human reference genome and, ultimately, an understanding of the genetic differences that make us human. SMRT assemblies and SV analyses. (Top) Contiguity of the de novo assemblies. (Bottom, left to right) For each ape, SVdetection was done against the human reference genome as represented by a dot plot of an inversion). Human-specific SVs, identified by comparing ape SVs and population genotyping (0/0, homozygous reference),were compared to single-cell gene expression differences [range: low (dark blue) to high (dark red)] in primary and organoid tissues. Each heatmap row is a gene that intersects an insertion or deletion (green), duplication (cyan), or inversion (light green). Genetic studies of human evolution require high-quality contiguous ape genome assemblies that are not guided by the human reference. We coupled long-read sequence assembly and full-length complementary DNA sequencing with a multiplatform scaffolding approach to produce ab initio chimpanzee and orangutan genome assemblies. By comparing these with two long-read de novo human genome assemblies and a gorilla genome assembly, we characterized lineage-specific and shared great ape genetic variation ranging from single– to mega–base pair–sized variants. We identified ~17,000 fixed human-specific structural variants identifying genic and putative regulatory changes that have emerged in humans since divergence from nonhuman apes. Interestingly, these variants are enriched near genes that are down-regulated in human compared to chimpanzee cerebral organoids, particularly in cells analogous to radial glial neural progenitors.

Orientation-dependent Dxz4 contacts shape the 3D structure of the inactive X chromosome

The mammalian inactive X chromosome (Xi) condenses into a bipartite structure with two superdomains of frequent long-range contacts, separated by a hinge region. Using Hi-C in edited mouse cells with allelic deletions or inversions within the hinge, here we show that the conserved Dxz4 locus is necessary to maintain this bipartite structure. Dxz4 orientation controls the distribution of contacts on the Xi, as shown by a massive reversal in long-range contacts after Dxz4 inversion. Despite an increase in CTCF binding and chromatin accessibility on the Xi in Dxz4-edited cells, only minor changes in TAD structure and gene expression were detected, in accordance with multiple epigenetic mechanisms ensuring X silencing. We propose that Dxz4 represents a structural platform for frequent long-range contacts with multiple loci in a direction dictated by the orientation of its bank of CTCF motifs, which may work as a ratchet to form the distinctive bipartite structure of the condensed Xi.The inactive X chromosome condenses into a bipartite structure. Here the authors use cells with allelic deletions or inversions to show that the Dxz4 locus is necessary to maintain the bipartite structure and that Dxz4 orientation controls the distribution of contacts on the inactive X chromosome.