Haloplex capture kit

Genomic DNA from either bone marrow fibroblasts, expanded peripheral blood T-cells, or whole blood cells was extracted. Comprehensive testing for 72 and 77 IBMFS genes respectively was done by an NGS IBMFS gene panel using the HaloPlex Capture Kit (Agilent Technologies, Santa Clara, CA) for DNA library preparation, and Illumina HiSeq 2500 platform for sequencing, as described previously.^{15 (link)} Sanger sequencing was done as previously described.^{13 (link)} Briefly, targeted genes were analyzed by bidirectional sequencing of individual exons and flanking intronic regions after PCR amplification.

Genomic DNA was extracted from peripheral blood, bone marrow fibroblasts, skin fibroblasts or expanded peripheral blood T-cells. Comprehensive NGS panel of known IBMFS genes was designed. Overall, 72, 77 and 141 genes, were included in the first, second, and third analyzed batch of patients, respectively (Supplementary Table 1). The Haloplex Capture Kit (Agilent Technologies, Santa Clara, CA) was used for DNA library preparation according to the manufacturer instructions. Briefly, the assay was based on a hybridization oligonucleotide pool, which covers coding regions, 50 bp flanking intronic regions that included splicing sites, 3′-untranslated regions that included potential translation regulatory elements, and upstream promoter regions. The oligonucleotides were 150 mers with 3× tiling and a maximum of 10 bp overlap between oligonucleotides. The panel design was submitted to the Agilent HaloPlex Design Wizard program (http://www.halogenomics.com/haloplex/custom-reagent-kits). Targeted fragments were amplified and were sequenced on the Illumina HiSeq2500 platform as previous described.^{6 (link)}