1. Targeted Next-Generation Sequencing for the Molecular Genetic Diagnostics of CardiomyopathiesClinical Perspective
by Benjamin Meder, Jan Haas, Andreas Keller, Christiane Heid, Steffen Just, Anne Borries, Valesca Boisguerin, Maren Scharfenberger-Schmeer, Peer Stähler;, Markus Beier, Dieter Weichenhan, Tim M. Strom, Arne Pfeufer, Bernhard Korn, Hugo A. Katus, and Wolfgang Rottbauer
Circ Genom Precis Med
Volume 4(2):
April 19, 2011
Copyright © American Heart Association, Inc. All rights reserved.

2. Targeted capturing of selected genomic fragments using hybridization-based microarrays.
Targeted capturing of selected genomic fragments using hybridization-based microarrays. Genomic DNA is sheared, fragment-ends are repaired, and sequencing adaptors for SOLiD NGS are attached. The genomic library is then hybridized to the oligonucleotide probes on a microfluidic array to bind specific DNA fragments. After washing away weakly bound unspecific fragments, the enriched library is eluted and transferred to NGS.
Benjamin Meder et al. Circ Cardiovasc Genet. 2011;4:
Copyright © American Heart Association, Inc. All rights reserved.

3. Sequence coverage of selected target genes.
Sequence coverage of selected target genes. Bar graphs representing the mean (black bars) ±SEM and median (gray bars) sequence coverage over the enriched target genes, ranging from 41 to 3083 in 10 consecutive patients with cardiomyopathy. The dashed line indicates the mean coverage (412) over all selected genes and patients.
Benjamin Meder et al. Circ Cardiovasc Genet. 2011;4:
Copyright © American Heart Association, Inc. All rights reserved.

4. Microarray-based target capturing leads to enrichment of selected genomic regions.
Microarray-based target capturing leads to enrichment of selected genomic regions. Shown is an example of the sequence coverage obtained for DES (depicted are exons 2 to 5) in 10 consecutive patients. Array-based enrichment aggregates sequence reads (blue) within the selected genomic regions, resulting in high sequence coverage of selected exons. Intronic regions, which were not enriched, are only poorly covered.
Benjamin Meder et al. Circ Cardiovasc Genet. 2011;4:
Copyright © American Heart Association, Inc. All rights reserved.

5. Enrichment of large disease genes.
Enrichment of large disease genes. A, Representative example of the sequence enrichment of the large TTN gene with ≈ bases coding the region. Shown are coding exons of TTN (yellow) and the corresponding sequence coverage (blue). B, Magnified view showing the selective enrichment of exons, whereas introns are only poorly covered.
Benjamin Meder et al. Circ Cardiovasc Genet. 2011;4:
Copyright © American Heart Association, Inc. All rights reserved.

6. Detection of a microdeletion in the MYBPC3 gene.
Detection of a microdeletion in the MYBPC3 gene. A, Overview of the sequence alignment against the reference sequence of MYBPC3. The rectangle indicates a deletion within MYBPC3. B, Magnified view of the rectangle showing a heterozygous microdeletion TT del within the coding region of MYBPC3, leading predictably to a frameshift starting at codon 412 (p.F412fs) and consecutively premature termination of protein translation. Because MYBPC3 is encoded on the reverse strand, the shown sequence reads are reverse complement. C, Sanger chromatogram showing the deletion and consecutive frameshift in MYBPC3 (forward read).
Benjamin Meder et al. Circ Cardiovasc Genet. 2011;4:
Copyright © American Heart Association, Inc. All rights reserved.

7. Nonsense mutations within MYH7 and LMNA
Nonsense mutations within MYH7 and LMNA. A, Sequence alignment of 50-bp SOLiD reads against the reference sequence of MYH7 (top).
Nonsense mutations within MYH7 and LMNA. A, Sequence alignment of 50-bp SOLiD reads against the reference sequence of MYH7 (top). In HCM patient ID3180, a missense mutation G428A in exon 5 of MYH7 predictably leads to the exchange of arginine with glutamine at position 143 (p.R143Q). A chromatogram confirms the heterozygote missense mutation (bottom). B, Sequence alignment showing a LMNA nonsense mutation in DCM patient ID3336, predictably leading to premature termination of protein translation (top). A chromatogram confirms the heterozygote stop mutation (bottom).
Benjamin Meder et al. Circ Cardiovasc Genet. 2011;4:
Copyright © American Heart Association, Inc. All rights reserved.

8. Identification of novel potential disease mutations.
Identification of novel potential disease mutations. A, In HCM patient ID3131, a heterozygous missense mutation, T484C, which was verified by Sanger sequencing (bottom), is predicted to lead to an amino acid exchange p.Y162H in MYH7. B, Sequence alignment against ILK gene. The nonsynonymous variant C209T was found in DCM patient ID3236, leading to the amino acid exchange p.P70L in ILK (bottom).
Benjamin Meder et al. Circ Cardiovasc Genet. 2011;4:
Copyright © American Heart Association, Inc. All rights reserved.