Probe Designs Are Very Different

There are four common filaggrin mutations in Scottish/Irishpopulations.Only NimbleGen is able to capture them all reliably.Nextera is misleading. Illumina do not publish their probesets,cannot know if mutations are well covered or not.

MethodologyFour patient samples had individual libraries made for each of thekits run in duplicate across two lanes per kit.The 24 datasets were aligned to the human genome (ensembl r71)with bowtie2 (v2.1.0) and had PCR duplicates removed with Picard(v1.89).Variant calling was performed with GATK (v2.2-8-g99996f2) usingvendor-provided probeset definitions and annotated with VariantEffect Predictor (v2.7).

Which is Best?

Samples were genotyped with Illumina OmniExpress Exome arrayand the results compared to the three WES platforms.15,068 positions common to genotype array and WES.Globally, Agilent and NimbleGen kits performed similarly with theIllumina kit being significantly worse.The Epidermal Differentiation Complex (EDC) region whichincludes 63 genes which are required for the normal developmentof the stratum corneum in skin.Within the EDC, NimbleGen has best probe coverage, best 20xcoverage and lowest disagreement with the genotyping arrayresults (Table 1).

AcknowledgementsWe thank the patients for providing the samples used in this study.

Exome SequencingSequencing libraries are prepared as for a normal genomic DNA

sample, but the DNA fragments are hydridised in solution to probesdesigned to enrich for coding regions in the genome. Non-codingDNA is washed away and captured fragments are eluted ready forsequencing on an Illumina HiSeq2000.

Quality Assessment

Agilent v4 shows bestbase coverage: 30x(largest circle).Agilent v5 shows worstduplicate rate: ~17%.Nextera & Agilent v5poor on-target rate: <50%NimbleGen shows goodon-target rate (~74%) andlow duplicate reads (~4%)

Variant Calling Reproducibility

Technical reproducibility ishigh with Agilent andNimbleGen (~91%), butNextera is worse: ~85%.Sample reproducibility isquite variable: 35-50%Background noise is high.

Sample Clustering

Samples cluster primarilyby kit.Protocol has more impacton results than biology.Clusters are robust asdetermined by bootstrapscores (au > 95%).

Christian Cole1,2, J Ward1,2,3, M Lee2,3, D Ross2,3, N wilson3, FJD Smith2, SJ Brown2, A Irvine4, WHI McLean2, GJ Barton1, and M Febrer3,

1Computational Biology, College of Life Sciences, University of Dundee, UK; 2Centre for Dermatology and Genetic Medicine, College of Life Sciences and College of Medicine, Dentistryand Nursing, University of Dundee, UK; 3Genomic Sequencing Unit, College of Life Sciences, University of Dundee, UK; 4Department of Dermatology, Our Lady’s Children’s Hospital,Dublin, Ireland

NOT ALL EXOMES ARE EQUAL:A COMPARISON OF THREE KITS

Aim

Whole exome sequencing as a protocol is highly dependent on the probedesign of the kit manufacturers. Here we present results from four humanpatient samples run against Illumina’s Nextera, Agilent’s SureSelect v5 andNimblegen’s SeqCap v3 library preparation kits sequenced across four lanesof a HiSeq2000. The data were processed through a variant calling pipelinebased around the Genome Analysis ToolKit (GATK). A comparison is madewith Illumina OmniExpressExome genotyping array data for validation. Thesignificant differences found have a particular relevance to dermatologyrelated studies which are an important focus for DGEM in Dundee, but alsoare more generally applicable to exome sequencing.

p.R501Xc.2285del4p.R2447Xp.S3247X

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Agilent SureSelect v4

Agilent SureSelect v5

Common Mutations in Atopic Eczema

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