Abstract 1327: Genome-wide loss-of-heterozygosity calling from low-pass sequencing of ligation-mediated whole genome amplified DNA in single tumor cells
Authors: Ferrarini Alberto , Forcato Claudio , Garonzi Marianna , Buson Genny , Pellicano Massimiliano , Fontana Francesca , Manaresi Nicolò 
Published: September 2020

Abstract
Background: Genome-wide analysis of Loss-of-heterozygosity (LoH) has been shown to be important in several contexts, including the assessment of BRCAness signature, a marker of homologous repair deficiency in several cancer types associated with efficacy of platinum therapy and PARP inhibitors. Methods to infer genome-wide LoH status require high-coverage whole-genome sequencing or low-pass sequencing of a large number of single cells. We present a method to infer genome-wide LoH status of as low as one single-cell from low-pass sequencing of libraries produced by Ampli1™ LowPass kit, at a fraction of the cost of current NGS-based methods.
Methods: We used low-pass sequencing data generated from 294 single Circulating Tumor Cells (CTCs) from prostate, multiple myeloma and lung cancer patients, and from 24 single cells from 8 cell lines. DNA amplified with Ampli1™ WGA kit (based on ligation-mediated PCR) combined with Ampli1 LowPass kit for Illumina generates a reduced genome representation, whereby a significant fraction of fragments is expected to be covered by more than one read. We reasoned that, even at a mean coverage insufficient for variant calling (≤0.05x), a statistically significant decrease in loci showing 2 alleles should be expected in LoH regions. A pileup of mapped sequences at dbSNP loci was obtained (minor allele frequency ≥5%; read depth ≥2). Mono- and bi-allelic loci (pileup containing 1 or 2 different alleles, respectively), were counted in each uniform copy-number (CN) genome segment within a single chromosome arm. Regions with CN equal to cell ploidy were used as internal reference. A one-sided Fisher test on biallelic sites depletion (multiplicity corrected p-value threshold of 0.05) was used to call LoH on each region.
Results: Compared with libraries obtained using random shearing, Ampli1 LowPass single-cell libraries showed an increase in mean coverage of covered genome from 1.1 to 1.9 starting from the same number of reads (1M) thus increasing the number of loci for LoH calling. Analysis of 10 patients' single cells with known LoH events, corresponding to regions at copy number 1, detected 1006 events out of 1142. Several (492) copy neutral LoH events or LoHs in copy gained regions were also detected. Representatives of such events were verified successfully by high pass sequencing of the same library. Performance was confirmed by analysis of 24 single cells from cell lines whose LoH status was inferred from CCLE database.
Conclusions: This innovative method leverages reduced genome representation inherent in Ampli1 LowPass libraries, to compute genome-wide LoH along with copy number (CN) status, meeting the need for LoH/CN analysis at single-cell level on rare-cells such as CTCs.
Citation Format: Alberto Ferrarini, Claudio Forcato, Marianna Garonzi, Genny Buson, Massimiliano Pellicano, Francesca Fontana, Nicolò Manaresi. Genome-wide loss-of-heterozygosity calling from low-pass sequencing of ligation-mediated whole genome amplified DNA in single tumor cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1327.