Circulating tumor cells (CTCs) enter peripheral blood from primary tumors and seed metastases. The genome sequencing of CTCs could offer noninvasive prognosis or even diagnosis, but has been hampered by low single-cell genome coverage of scarce CTCs. Here, we report the use of the recently developed multiple annealing and looping-based amplification cycles for whole-genome amplification of single CTCs from lung cancer patients. We observed characteristic cancer-associated single-nucleotide variations and insertions/deletions in exomes of CTCs. These mutations provided information needed for individualized therapy, such as drug resistance and phenotypic transition, but were heterogeneous from cell to cell. In contrast, every CTC from an individual patient, regardless of the cancer subtypes, exhibited reproducible copy number variation (CNV) patterns, similar to those of the metastatic tumor of the same patient. Interestingly, different patients with the same lung cancer adenocarcinoma (ADC) shared similar CNV patterns in their CTCs. Even more interestingly, patients of smallcell lung cancer have CNV patterns distinctly different from those of ADC patients. Our finding suggests that CNVs at certain genomic loci are selected for the metastasis of cancer. The reproducibility of cancer-specific CNVs offers potential for CTC-based cancer diagnostics.cancer diagnostics | personalized therapy A s a genomic disease, cancer involves a series of changes in the genome, starting from primary tumors, via circulating tumor cells (CTCs), to metastases that cause the majority of mortalities (1-3). These genomic alterations include copy number variations (CNVs), single-nucleotide variations (SNVs), and insertions/deletions (INDELs). Regardless of the concentrated efforts in the past decades, the key driving genomic alterations responsible for metastases are still elusive (1).For noninvasive prognosis and diagnosis of cancer, it is desirable to monitor genomic alterations through the circulatory system. Genetic analyses of cell-free DNA fragments in peripheral blood have been reported (4-6) and recently extended to the whole-genome scale (7-9). However, it may be advantageous to analyze CTCs, as they represent intact functional cancer cells circulating in peripheral blood (10). Although previous studies have shown that CTC counting was able to predict progression and overall survival of cancer patients (11,12), genomic analyses of CTCs could provide more pertinent information for personalized therapy (13). However, it is difficult to probe the genomic changes in DNA obtainable from the small number of captured CTCs. To meet this challenge, a single-cell whole-genome amplification (WGA) method, multiple annealing and loopingbased amplification cycles (MALBAC) (14), has been developed to improve the amplification uniformity across the entire genome over previous methods (15,16), allowing precise determination of CNVs and detection of SNVs with a low false-positive rate in a single cell. Here, we present genomic analyses of CTCs from...

Meiotic recombination creates genetic diversity and ensures segregation of homologous chromosomes. Previous population analyses yielded results averaged among individuals and impacted by evolutionary pressures. Here we sequenced 99 sperm from an Asian male using the newly developed amplification method—Multiple Annealing and Looping-Based Amplification Cycles (MALBAC)—to phase the personal genome and map at high resolution recombination events, which are non-uniformly distributed across the genome in the absence of selection pressure. The paucity of recombination near transcription start sites observed in individual sperm indicates such a phenomenon is intrinsic to the molecular mechanism of meiosis. Interestingly, a decreased crossover frequency in companion with an increase of autosomal aneuploidy is observable on a global per-sperm basis.

Single-cell genome analyses of human oocytes are important for meiosis research and preimplantation genomic screening. However, the nonuniformity of single-cell whole-genome amplification hindered its use. Here, we demonstrate genome analyses of single human oocytes using multiple annealing and looping-based amplification cycle (MALBAC)-based sequencing technology. By sequencing the triads of the first and second polar bodies (PB1 and PB2) and the oocyte pronuclei from same female egg donors, we phase the genomes of these donors with detected SNPs and determine the crossover maps of their oocytes. Our data exhibit an expected crossover interference and indicate a weak chromatid interference. Further, the genome of the oocyte pronucleus, including information regarding aneuploidy and SNPs in disease-associated alleles, can be accurately deduced from the genomes of PB1 and PB2. The MALBAC-based preimplantation genomic screening in in vitro fertilization (IVF) enables accurate and cost-effective selection of normal fertilized eggs for embryo transfer.

Tumor-derived cell-free DNA (cfDNA) has biomarker potential; therefore, this study aimed to identify cfDNA in the aqueous humor (AH) of retinoblastoma eyes and correlate somatic chromosomal copy-number alterations (SCNA) with clinical outcomes, specifically eye salvage. AH was extracted via paracentesis during intravitreal injection of chemotherapy or enucleation. Shallow whole-genome sequencing was performed using isolated cfDNA to assess for highly recurrent SCNAs in retinoblastoma including gain of 1q, 2p, 6p, loss of 13q, 16q, and focal amplification. Sixty-three clinical specimens of AH from 29 eyes of 26 patients were evaluated; 13 eyes were enucleated and 16 were salvaged (e.g., saved). The presence of detectable SCNAs was 92% in enucleated eyes versus 38% in salvaged eyes ( = 0.006). Gain of chromosome 6p was the most common SCNA found in 77% of enucleated eyes, compared with 25% of salvaged eyes ( = 0.0092), and associated with a 10-fold increased odds of enucleation (OR, 10; 95% CI, 1.8-55.6). The median amplitude of 6p gain was 1.47 in enucleated versus 1.07 in salvaged eyes ( = 0.001). The presence of AH SCNAs was correlated retrospectively with eye salvage. The probability of ocular salvage was higher in eyes without detectable SCNAs in the AH ( = 0.0028), specifically 6p gain. This is the first study to correlate clinical outcomes with SCNAs in the AH from retinoblastoma eyes, as such these findings indicate that 6p gain in the aqueous humor is a potential prognostic biomarker for poor clinical response to therapy. The correlation of clinical outcomes and SCNAs in the AH identified in the current study requires prospective studies to validate these finding before SCNAs, like 6p gain, can be used to predict clinical outcomes at diagnosis. .

IMPORTANCE Retinoblastoma (Rb) is one of the first tumors to have a known genetic etiology. However, because biopsy of this tumor is contraindicated, it has not been possible to define the effects of secondary genetic changes on the disease course. OBJECTIVE To investigate whether the aqueous humor (AH) of Rb eyes has sufficient tumor-derived DNA to perform genetic analysis of the tumor, including DNA copy number alterations. DESIGN, SETTING, AND PARTICIPANTS This investigation was a case series study at a tertiary care hospital (Children's Hospital Los Angeles) with a large Rb treatment center. Cell-free DNA (cfDNA) was isolated from 6 AH samples from 3 children with Rb, including 2 after primary enucleation and 1 undergoing multiple intravitreous injections of melphalan for vitreous seeding. Samples were taken between December 2014 and September 2015. MAIN OUTCOMES AND MEASURES Measurable levels of nucleic acids in the AH and identification of tumor-derived DNA copy number variation in the AH. The AH was analyzed for DNA, RNA, and micro-RNA using Qubit high-sensitivity kits. Cell-free DNA was isolated from the AH, and sequencing library protocols were optimized. Shallow whole-genome sequencing was performed on an Illumina platform, followed by genome-wide chromosomal copy number variation profiling to assess the presence of tumor DNA fractions in the AH cfDNA of the 3 patients. One child's cfDNA from the AH and tumor DNA were subjected to Sanger sequencing to isolate the RB1 mutation. RESULTS Six AH samples were obtained from 3 Rb eyes in 3 children (2 male and 1 female; diagnosed at ages 7, 20, and 28 months). A corroborative pattern between the chromosomal copy number variation profiles of the AH cfDNA and tumor-derived DNA from the enucleated samples was identified. In addition, a nonsense RB1 mutation (Lys→STOP) from 1 child was also identified from the AH samples obtained during intravitreous injection of melphalan, which matched the tumor sample postsecondary enucleation. Sanger sequencing of the AH cfDNA and tumor DNA with polymerase chain reaction primers targeting RB1 gene c.1075A demonstrated this same RB1 mutation. CONCLUSIONS AND RELEVANCE In this study evaluating nucleic acids in the AH from Rb eyes undergoing salvage therapy with intravitreous injection of melphalan, the results suggest that the AH can serve as a surrogate tumor biopsy when Rb tumor tissue is not available. This novel method will allow for analyses of tumor-derived DNA in Rb eyes undergoing salvage therapy that have not been enucleated.