Daniel Backenroth1, Fouad Zahdeh2, Yehuda Kling2, Aharon Peretz3, Tzvia Rosen2, Dina Kort2, Sharon Zeligson4, Tal Dror4, Sophie Kirshberg4, Efrat Burak4, Reeval Segel4,5, Ephrat Levy-Lahad4,5, David Zangen6, Gheona Altarescu4,5, Shai Carmi7, David A Zeevi8. 1. Braun School of Public Health and Community Medicine, The Hebrew University of Jerusalem, Jerusalem, Israel. 2. Translational Genomics Lab, Medical Genetics Institute, Shaare Zedek Medical Center, Jerusalem, Israel. 3. IVF Unit, Division of Obstetrics and Gynecology, Shaare Zedek Medical Center, Jerusalem, Israel. 4. Medical Genetics Institute, Shaare Zedek Medical Center, Jerusalem, Israel. 5. Hebrew University Faculty of Medicine, Jerusalem, Israel. 6. Division of Pediatric Endocrinology and Diabetes, Hadassah Hebrew University Medical Center, Jerusalem, Israel. 7. Braun School of Public Health and Community Medicine, The Hebrew University of Jerusalem, Jerusalem, Israel. shai.carmi@mail.huji.ac.il. 8. Translational Genomics Lab, Medical Genetics Institute, Shaare Zedek Medical Center, Jerusalem, Israel. zeevidavid@szmc.org.il.
Abstract
PURPOSE: To develop an economical, user-friendly, and accurate all-in-one next-generation sequencing (NGS)-based workflow for single-cell gene variant detection combined with comprehensive chromosome screening in a 24-hour workflow protocol. METHODS: We subjected single lymphoblast cells or blastomere/blastocyst biopsies from four different families to low coverage (0.3×-1.4×) genome sequencing. We combined copy-number variant (CNV) detection and whole-genome haplotype phase prediction via Haploseek, a novel, user-friendly analysis pipeline. We validated haplotype predictions for each sample by comparing with clinical preimplantation genetic diagnosis (PGD) case results or by single-nucleotide polymorphism (SNP) microarray analysis of bulk DNA from each respective lymphoblast culture donor. CNV predictions were validated by established commercial kits for single-cell CNV prediction. RESULTS: Haplotype phasing of the single lymphoblast/embryo biopsy sequencing data was highly concordant with relevant ground truth haplotypes in all samples/biopsies from all four families. In addition, whole-genome copy-number assessments were concordant with the results of a commercial kit. CONCLUSION: Our results demonstrate the establishment of a reliable method for all-in-one molecular and chromosomal diagnosis of single cells. Important features of the Haploseek pipeline include rapid sample processing, rapid sequencing, streamlined analysis, and user-friendly reporting, so as to expedite clinical PGD implementation.
PURPOSE: To develop an economical, user-friendly, and accurate all-in-one next-generation sequencing (NGS)-based workflow for single-cell gene variant detection combined with comprehensive chromosome screening in a 24-hour workflow protocol. METHODS: We subjected single lymphoblast cells or blastomere/blastocyst biopsies from four different families to low coverage (0.3×-1.4×) genome sequencing. We combined copy-number variant (CNV) detection and whole-genome haplotype phase prediction via Haploseek, a novel, user-friendly analysis pipeline. We validated haplotype predictions for each sample by comparing with clinical preimplantation genetic diagnosis (PGD) case results or by single-nucleotide polymorphism (SNP) microarray analysis of bulk DNA from each respective lymphoblast culture donor. CNV predictions were validated by established commercial kits for single-cell CNV prediction. RESULTS: Haplotype phasing of the single lymphoblast/embryo biopsy sequencing data was highly concordant with relevant ground truth haplotypes in all samples/biopsies from all four families. In addition, whole-genome copy-number assessments were concordant with the results of a commercial kit. CONCLUSION: Our results demonstrate the establishment of a reliable method for all-in-one molecular and chromosomal diagnosis of single cells. Important features of the Haploseek pipeline include rapid sample processing, rapid sequencing, streamlined analysis, and user-friendly reporting, so as to expedite clinical PGD implementation.