Distribution patterns of segmental aneuploidies in human blastocysts identified by next- generation sequencing  María Vera-Rodríguez, M.Sc., Claude-Edouard Michel, Ph.D., Amparo Mercader, Ph.D., Alex J. Bladon, M.Phys., Ph.D., Lorena Rodrigo, Ph.D., Felix Kokocinski, Ph.D., Emilia Mateu, Ph.D., Nasser Al-Asmar, MSc, David Blesa, PhD, Carlos Simón, MD, Ph.D., Carmen Rubio, Ph.D.  Fertility and Sterility  Volume 105, Issue 4, Pages 1047-1055.e2 (April 2016) DOI: 10.1016/j.fertnstert.2015.12.022 Copyright © 2016 American Society for Reproductive Medicine Terms and Conditions

Figure 1 Study design for the analysis of concordance rates between next-generation sequencing (NGS) and array comparative genomic hybridization (aCGH). Number of samples and events included in the study, divided between the control group (samples from translocations carriers) and study group (samples from couples with normal karyotype). Number of events from both groups reanalyzed by fluorescent in situ hybridization (FISH) of individual blastocyst cells. Fertility and Sterility 2016 105, 1047-1055.e2DOI: (10.1016/j.fertnstert.2015.12.022) Copyright © 2016 American Society for Reproductive Medicine Terms and Conditions

Figure 2 Concordance rates between next-generation sequencing (NGS), array comparative genomic hybridization (aCGH), and fluorescent in situ hybridization (FISH) for aneuploidy detection. (A) The concordance rates were calculated per analyzed chromosome, including euploid and aneuploidy chromosomes, and exclusively per aneuploid chromosome. Each type of concordance rate was calculated for all aneuploidies, for whole-chromosome aneuploidies and for segmental aneuploidies. Finally, the concordance rates for segmental aneuploidies were calculated for those events detected in the control group (translocations carriers) and the study group (normal karyotypes). (B) The concordance rates for those samples with FISH analyses were calculated per aneuploidy chromosome as all events analyzed were previously detected as segmental aneuploidies by aCGH and NGS. The concordance rates were also differentiated between the control and the study group, and between pure and mosaic events inside the study group. Fertility and Sterility 2016 105, 1047-1055.e2DOI: (10.1016/j.fertnstert.2015.12.022) Copyright © 2016 American Society for Reproductive Medicine Terms and Conditions

Figure 3 Comparison of segmental aneuploidy detection by array comparative genomic hybridization (aCGH) and next-generation sequencing (NGS). (A) Both aCGH and NGS detected similarly a pure segmental aneuploidy for chromosome 6. (B) One segmental aneuploidy for chromosome 20 identified as pure by aCGH was detected by NGS as a mosaic pattern. (C) Array CGH and NGS detected an aneuploidy for chromosome 3 showing a mosaic pattern in both cases. (D) One mosaic segmental aneuploidy for chromosome 8 detected by NGS was not originally detected by aCGH. Fertility and Sterility 2016 105, 1047-1055.e2DOI: (10.1016/j.fertnstert.2015.12.022) Copyright © 2016 American Society for Reproductive Medicine Terms and Conditions

Figure 4 Segmental aneuploidies analyzed by array comparative genomic hybridization (aCGH), next-generation sequencing (NGS), and fluorescent in situ hybridization (FISH). Aneuploidies were identified as pure if they scored beyond the euploidy confidence range in aCGH and NGS, or if all cells analyzed by FISH were aneuploid. Aneuploidies were identified as mosaic if they showed an intermediate value within the confidence range, or, in FISH analysis, if aneuploid and euploid cells were detected in the same embryo. The color scale represents the percentage of aneuploid cells in the group of cells analyzed by FISH, with dark green indicating 100% aneuploidy, dark red indicating 0 aneuploidy, and intermediary colors indicating different levels of mosaicism (see Supplemental Fig. 2). Fertility and Sterility 2016 105, 1047-1055.e2DOI: (10.1016/j.fertnstert.2015.12.022) Copyright © 2016 American Society for Reproductive Medicine Terms and Conditions

Supplemental Figure 1 Comparison of array comparative genomic hybridization (aCGH) and next-generation sequencing (NGS) whole-chromosome aneuploidy detection. (A) A pure, whole-chromosome aneuploidy detected by aCGH for chromosome 9 was also detected by NGS as pure. (B) A mosaic whole-chromosome aneuploidy detected by aCGH for chromosome 18 showed a similar pattern using NGS. (C) NGS detected mosaic aneuploidies for chromosomes 12 and 16 that were not initially identified by aCGH. Fertility and Sterility 2016 105, 1047-1055.e2DOI: (10.1016/j.fertnstert.2015.12.022) Copyright © 2016 American Society for Reproductive Medicine Terms and Conditions

Supplemental Figure 2 Fluorescent in situ hybridization (FISH) analysis for the detection of segmental aneuploidies in blastocyst cells. (A–C) FISH analysis of blastocyst in which a segmental loss (55.3 Mb) in the p arm of chromosome 1 was previously detected by array comparative genomic hybridization (aCGH) and next-generation sequencing (NGS) showing a mosaic pattern. We analyzed 34 cells successfully using Spectrum red probes specific to the p subtelomere region of chromosome 1 (Aquarius; Cytocell), with (A) 12 cells (23.5%) containing a single signal, (B) eight cells (35.3%) with two signals, and (C) 14 cells (41.2%) with three signals. Fertility and Sterility 2016 105, 1047-1055.e2DOI: (10.1016/j.fertnstert.2015.12.022) Copyright © 2016 American Society for Reproductive Medicine Terms and Conditions