A Single Cell Level Based Method for Copy Number Variation Analysis by Low Coverage Massively Parallel Sequencing

Abstract

Copy number variations (CNVs), a common genomic mutation associated with various diseases, are important in research and clinical applications. Whole genome amplification (WGA) and massively parallel sequencing have been applied to single cell CNVs analysis, which provides new insight for the fields of biology and medicine. However, the WGA-induced bias significantly limits sensitivity and specificity for CNVs detection. Addressing these limitations, we developed a practical bioinformatic methodology for CNVs detection at the single cell level using low coverage massively parallel sequencing. This method consists of GC correction for WGA-induced bias removal, binary segmentation algorithm for locating CNVs breakpoints, and dynamic threshold determination for final signals filtering. Afterwards, we evaluated our method with seven test samples using low coverage sequencing (4∼9.5%). Four single-cell samples from peripheral blood, whose karyotypes were confirmed by whole genome sequencing analysis, were acquired. Three other test samples derived from blastocysts whose karyotypes were confirmed by SNP-array analysis were also recruited. The detection results for CNVs of larger than 1 Mb were highly consistent with confirmed results reaching 99.63% sensitivity and 97.71% specificity at base-pair level. Our study demonstrates the potential to overcome WGA-bias and to detect CNVs (>1 Mb) at the single cell level through low coverage massively parallel sequencing. It highlights the potential for CNVs research on single cells or limited DNA samples and may prove as a promising tool for research and clinical applications, such as pre-implantation genetic diagnosis/screening, fetal nucleated red blood cells research and cancer heterogeneity analysis.

Clinical utilisation of a rapid low-pass whole genome sequencing technique for the diagnosis of aneuploidy in human embryos prior to implantation

“…The results of the current research confirm that NGS provides highly accurate aneuploidy detection at substantially lower cost than existing methods used for PGS and that testing can be performed within a fresh IVF cycle, avoiding the complication of cryopreservation. A small number of previous studies had suggested that the combination of WGA and NGS might be capable of producing useful genomic data from single cells 12––15. However, previous attempts to harness massively-parallel sequencing methods for the detection of aneuploidy in single cells required several days to complete, meaning that preimplantation genetic testing could only be carried out if embryos were cryopreserved after biopsy.…”

Advances and Applications of Single-Cell Sequencing Technologies

“…This processing pipeline was developed into a user-friendly web server platform with impressive visualization tools called Ginkgo (http://gb.cshl.edu/ginkgo). Another copy number method uses SCS read count data generated from DOP-PCR that corrects for GC bias and performs binary segmentation followed by dynamic thresholding (Zhang et al, 2013). …”

Embryo Genome Profiling by Single-Cell Sequencing for Preimplantation Genetic Diagnosis in a β-Thalassemia Family

“…1A). We successfully phased 131 785 and 133 601 of the maternal and paternal heterozygous sites, including mutations in the hemoglo- bin, beta (HBB) 14 gene. Subsequently, we used an HMM to determine the parental transmission, identifying recombination breakpoints and filtering out sequencing error at the same time.…”

“…Concomitantly, the accuracy of the final genotypes stabilized above 97%. This in- 14 Human genes: HBB, hemoglobin, beta; HLA-DRB1, SlC25A22, solute carrier family 25 (mitochondrial carrier: glutamate), member 22; INS, insulin; SMPD1, sphingomyelin phosphodiesterase 1, acid lysosomal; TH, tyrosine hydroxylase.…”

“…Karyomapping based on single-nucleotide polymorphism (SNP) array has been reported to overcome the ADO obstacles in single-cell analysis (10 ). For the embryonic aneuploidy testing, detection of chromosomal abnormalities based on array comparative genomic hybridization (11 ), SNP array (12 ), or next-generation sequencing (NGS) (13)(14)(15)(16)(17) in biopsied embryonic cells have been published. In this study, genome profiling of human embryos using single blastomere whole-genome sequencing (WGS) was assessed in a ␤-thalassemia family.…”