Jennifer Troge

Tumour evolution inferred by single-cell sequencing

Genomic analysis provides insights into the role of copy number variation in disease, but most methods are not designed to resolve mixed populations of cells. In tumours, where genetic heterogeneity is common, very important information may be lost that would be useful for reconstructing evolutionary history. Here we show that with flow-sorted nuclei, whole genome amplification and next generation sequencing we can accurately quantify genomic copy number within an individual nucleus. We apply single-nucleus sequencing to investigate tumour population structure and evolution in two human breast cancer cases. Analysis of 100 single cells from a polygenomic tumour revealed three distinct clonal subpopulations that probably represent sequential clonal expansions. Additional analysis of 100 single cells from a monogenomic primary tumour and its liver metastasis indicated that a single clonal expansion formed the primary tumour and seeded the metastasis. In both primary tumours, we also identified an unexpectedly abundant subpopulation of genetically diverse ‘pseudodiploid’ cells that do not travel to the metastatic site. In contrast to gradual models of tumour progression, our data indicate that tumours grow by punctuated clonal expansions with few persistent intermediates.

The contribution of de novo coding mutations to autism spectrum disorder

Whole exome sequencing has proven to be a powerful tool for understanding the genetic architecture of human disease. Here we apply it to more than 2,500 simplex families, each having a child with an autistic spectrum disorder. By comparing affected to unaffected siblings, we show that 13% of de novo missense mutations and 43% of de novo likely gene-disrupting (LGD) mutations contribute to 12% and 9% of diagnoses, respectively. Including copy number variants, coding de novo mutations contribute to about 30% of all simplex and 45% of female diagnoses. Almost all LGD mutations occur opposite wild-type alleles. LGD targets in affected females significantly overlap the targets in males of lower intelligence quotient (IQ), but neither overlaps significantly with targets in males of higher IQ. We estimate that LGD mutation in about 400 genes can contribute to the joint class of affected females and males of lower IQ, with an overlapping and similar number of genes vulnerable to contributory missense mutation. LGD targets in the joint class overlap with published targets for intellectual disability and schizophrenia, and are enriched for chromatin modifiers, FMRP-associated genes and embryonically expressed genes. Most of the significance for the latter comes from affected females.

Inferring tumor progression from genomic heterogeneity

Cancer progression in humans is difficult to infer because we do not routinely sample patients at multiple stages of their disease. However, heterogeneous breast tumors provide a unique opportunity to study human tumor progression because they still contain evidence of early and intermediate subpopulations in the form of the phylogenetic relationships. We have developed a method we call Sector-Ploidy-Profiling (SPP) to study the clonal composition of breast tumors. SPP involves macro-dissecting tumors, flow-sorting genomic subpopulations by DNA content, and profiling genomes using comparative genomic hybridization (CGH). Breast carcinomas display two classes of genomic structural variation: (1) monogenomic and (2) polygenomic. Monogenomic tumors appear to contain a single major clonal subpopulation with a highly stable chromosome structure. Polygenomic tumors contain multiple clonal tumor subpopulations, which may occupy the same sectors, or separate anatomic locations. In polygenomic tumors, we show that heterogeneity can be ascribed to a few clonal subpopulations, rather than a series of gradual intermediates. By comparing multiple subpopulations from different anatomic locations, we have inferred pathways of cancer progression and the organization of tumor growth.

Genome-wide copy number analysis of single cells

Copy number variation (CNV) is increasingly recognized as an important contributor to phenotypic variation in health and disease. Most methods for determining CNV rely on admixtures of cells in which information regarding genetic heterogeneity is lost. Here we present a protocol that allows for the genome-wide copy number analysis of single nuclei isolated from mixed populations of cells. Single-nucleus sequencing (SNS), combines flow sorting of single nuclei on the basis of DNA content and whole-genome amplification (WGA); this is followed by next-generation sequencing to quantize genomic intervals in a genome-wide manner. Multiplexing of single cells is discussed. In addition, we outline informatic approaches that correct for biases inherent in the WGA procedure and allow for accurate determination of copy number profiles. All together, the protocol takes ∼3 d from flow cytometry to sequence-ready DNA libraries.

Novel genomic alterations and clonal evolution in chronic lymphocytic leukemia revealed by representational oligonucleotide microarray analysis (ROMA)

We examined copy number changes in the genomes of B cells from 58 patients with chronic lymphocytic leukemia (CLL) by using representational oligonucleotide microarray analysis (ROMA), a form of comparative genomic hybridization (CGH), at a resolution exceeding previously published studies. We observed at least 1 genomic lesion in each CLL sample and considerable variation in the number of abnormalities from case to case. Virtually all abnormalities previously reported also were observed here, most of which were indeed highly recurrent. We observed the boundaries of known events with greater clarity and identified previously undescribed lesions, some of which were recurrent. We profiled the genomes of CLL cells separated by the surface marker CD38 and found evidence of distinct subclones of CLL within the same patient. We discuss the potential applications of high-resolution CGH analysis in a clinical setting.

Application of ROMA (representational oligonucleotide microarray analysis) to patients with cytogenetic rearrangements

Purpose: To demonstrate the accuracy and sensitivity of Representational Oligonucleotide Microarray Analysis (ROMA) to describe copy number changes in patients with chromosomal abnormalities.Methods: ROMA was performed using BglII digested DNA from two cases with cytogenetically detected deletions and one case with an unbalanced terminal rearrangement detected only by subtelomeric FISH. Hybridization was to an 85,000-probe oligonucleotide microarray, providing an average resolution of 35 kb. FISH was used to confirm some of the ROMA findings.Results: By ROMA, a del(13)(q14.3q21.2) was shown to be noncontiguous, with deletions extending from 53.08 to 61.40 Mb and from 72.88 to 74.83 Mb. The 10-Mb deletion contained only six known genes. FISH confirmed the noncontiguous nature of the deletion, as well as a small amplification in 6q that was also found in the patients mother. A del(4)(q12q21.2) was found by ROMA to be 23 Mb in length, from 58.8 to 81.9 Mb on chromosome 4, in agreement with the cytogenetically assigned breakpoints. ROMA showed that an unbalanced “subtelomeric” rearrangement involved a 6-Mb deletion of 22q and an 8-Mb duplication of 16q.Conclusions: ROMA can define cytogenetic aberrations with extraordinary precision. Unexpected findings included the interrupted nature of the deletion in 13q and the large size of the imbalances in the “subtelomeric” rearrangement. Together with the information from the human genome sequence and proteomics, the ability to define rearrangements with “ultra-high” resolution will improve the ability to provide accurate prognosis both prenatally and postnatally to parents of offspring with chromosomal aberrations.

PROBER: oligonucleotide FISH probe design software

UNLABELLED PROBER is an oligonucleotide primer design software application that designs multiple primer pairs for generating PCR probes useful for fluorescence in situ hybridization (FISH). PROBER generates Tiling Oligonucleotide Probes (TOPs) by masking repetitive genomic sequences and delineating essentially unique regions that can be amplified to yield small (100-2000 bp) DNA probes that in aggregate will generate a single, strong fluorescent signal for regions as small as a single gene. TOPs are an alternative to bacterial artificial chromosomes (BACs) that are commonly used for FISH but may be unstable, unavailable, chimeric, or non-specific to small (10-100 kb) genomic regions. PROBER can be applied to any genomic locus, with the limitation that the locus must contain at least 10 kb of essentially unique blocks. To test the software, we designed a number of probes for genomic amplifications and hemizygous deletions that were initially detected by Representational Oligonucleotide Microarray Analysis of breast cancer tumors. AVAILABILITY http://prober.cshl.edu

Corrigendum: Genome-wide copy number analysis of single cells.

Nat. Protoc. 7, 1024–1041 (2012); published online 3 May 2012; corrected after print 24 February 2016 In the version of this article initially published, the units for the concentration of NaCl in the NST buffer described in the Reagent Setup section were incorrect. The correct unit should be mM. The error has been corrected in the HTML and PDF versions of the article.