Implementation of a Reliable Next-Generation Sequencing Strategy for Molecular Diagnosis of Dystrophinopathies  Melissa Alame, Delphine Lacourt, Reda Zenagui, Déborah Mechin, Fabienne Danton, Michel Koenig, Mireille Claustres, Mireille Cossée  The Journal of Molecular Diagnostics  Volume 18, Issue 5, Pages 731-740 (September 2016) DOI: 10.1016/j.jmoldx.2016.05.003 Copyright © 2016 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 1 Optimization of the DMD MASTR kit (Multiplicom). A: Coverage comparison of two DMD MASTR kit designs after NGS on a GS-Junior sequencer (Roche). The first design contains 105 amplicons but does not allow coverage of all 79 exons and exons-intron boundaries >40 reads each with the GS-Junior sequencer. Three percent of the sequences are not covered properly. A new version of the DMD MASTR kit (v2) has been designed, containing 118 amplicons that allow a coverage >40× for all of the sequences except for 1 amplicon of the 708 (99.8%) amplicons. One hundred percent of amplicons have read counts >30×. B: Comparison of the coverage results for Roche (GS-Junior) and Illumina (MiSeq) technologies. Amplicons have a coverage much higher with MiSeq sequencer than with GS-Junior sequencer, due to the higher sequencing capacity of the MiSeq sequencer. C: Quality score of bases obtained with MiSeq sequencer (Illumina). The horizontal scale represents the Q-score and the vertical scale the total number of reads. The histogram shows that 81.7% of the reads have a Q-score >30 in our experiment, which correspond to good quality sequencing. DMD, Duchenne muscular dystrophy; MASTR, Multiplex Amplification of Specific Targets for Resequencing; NGS, next-generation sequencing; Q-score, quality score. The Journal of Molecular Diagnostics 2016 18, 731-740DOI: (10.1016/j.jmoldx.2016.05.003) Copyright © 2016 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 2 Improvement of the quality results within homopolymeric sequences using MiSeq sequencer. Multiplicom library from a DNA with a deletion of four bases in the DMD gene, c.10554-36_10554-33delCTTT (intron 74), was sequenced using a GS-Junior and a MiSeq sequencer. Results of forward sequencing analyzed by SeqNext were compared. Deletions are shown as rectangles with arrows at both ends pointing downward above the pseudo-electrophoregram. Arrows in red rectangles correspond to deletions classified in the distinct table, arrow in gray rectangles correspond to deletions not classified in the distinct table. In front of the proximal arrow is shown the allelic frequency of each deletion. In the reads sequences, deletions are marked by a minus sign (−) highlighted in blue. GS-Junior sequencing: The correct deletion c.10554-36_10554-33delCTTT is detected in 30.1% of the reads, incorrect deletions are identified in 50.7% of reads. MiSeq sequencing: The correct deletion c.10554-36_10554-33delCTTT is detected in 81.2% of the reads and an incorrect deletion (+1 deleted base) in 12.5% of reads. The Journal of Molecular Diagnostics 2016 18, 731-740DOI: (10.1016/j.jmoldx.2016.05.003) Copyright © 2016 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 3 A: Representation of CNV detection by the CNV SeqNext software version 3.5.0. A complex rearrangement (exon 45 to 47 and exon 53 to 55 hemizygous duplication) was identified by our pipeline. For each amplicon, the control RPC (represented by a purple histogram) is compared with the patient RPC (represented by a green histogram) in SeqNext software (module CNV). The RPC ratio for the patient DNA is represented by the blue histogram (Material and Methods). The ratios thresholds for normal copy numbers are represented by two red lines. The RPC ratios for exons 45, 46, 47, 53, 54, and 55 overpass 130% and no false positives are observed. B: RPC ratio average of the different CNV types. The histogram represents a comparison of the RPC ratios obtained with 7 DNA libraries (light gray) versus 12 (dark gray) DNA libraries sequenced simultaneously on a nano flow cell, and theoretical ratios (white) for absence of CNV, heterozygous deletions, heterozygous duplications, and hemizygous duplications. The means, SDs, and ranges of RPC ratios are represented in Table 4. It shows that the RPC ratios obtained for the different CNV types are closed to the theoretical ratios. The variability seems to be less important when pooling seven DNA libraries. CNV, copy number variation; RPC, relative PCR coverage. The Journal of Molecular Diagnostics 2016 18, 731-740DOI: (10.1016/j.jmoldx.2016.05.003) Copyright © 2016 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 4 New molecular diagnosis strategy after the implementation of NGS. The DMD MASTR kit with MiSeq sequencing technology will be used to screen CNVs and SNVs in the 79 exons and exon-intron junctions of the DMD gene of a patient’s DNA. If NGS technology cannot detect any mutations and if muscular biopsy is available, molecular RNA analysis will be performed to search for deep intronic mutations affecting normal splicing of the dystrophin mRNA. CNV, copy number variation; DMD, Duchenne muscular dystrophy; MASTR, Multiplex Amplification of Specific Targets for Resequencing; NGS, next-generation sequencing; SNV, single nucleotide variation. The Journal of Molecular Diagnostics 2016 18, 731-740DOI: (10.1016/j.jmoldx.2016.05.003) Copyright © 2016 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions