2.3 Mutation screening
The genomic DNA (50 ng) of each sample was enzyme-digested into around 200bp of fragments. The DNA fragments were end-repaired (the 3’end was added one adenine base) and were ligated with barcoded sequencing adaptors. And the ligated fragments about 320bp were captured by XP beads. After PCR amplification, the DNA fragments were hybridized by Nano WES according to the manufacturer’s Protocol. The hybridized DNA products were eluted and then subjected to PCR amplification and purification as a DNA library for each sample. Next, the libraries were quantified by qPCR and size distribution were determined using Nano WES (Berry Genomics, China). Finally, Novaseq6000 platform (Illumina, San Diego, USA, with 150 bp pair-end sequencing mode) was used for genomic DNA sequencing. Raw image files were processed using CASAVA v1.82 for base calling and generating raw data. The sequencing reads were aligned to the human reference genome (hg19/GRCh37) using Burrows–Wheeler Aligner tool and PCR duplicates were removed using Picard v1.57 (http://picard.sourceforge.net/). Verita Trekker® Variants Detection System by Berry Genomics and the third-party software GATK (https://software.broadinstitute.org/gatk/) was employed for variant calling. Variant annotation and interpretation were conducted by ANNOVAR (Wang, et al, 2010) and the Enliven® Variants Annotation Interpretation System (a comprehensive tool called Sprinkle was developed and authorized by Berry Genomics) was used for CNV calling. It includes XHMM PCA method (sequencing noise removal), CNV Kit fix module (GC and bias correction), and copy number calculation. The identification of CNV were performed in exons and long segment areas.
Mutation Surveyor Demo software version 4.0 was used to analyze the sample sequences comparing with the reference sequences from the National Center for Biotechnology Information (NCBI) (EXT1 : NM_ 000127.2; EXT2 : NM_000401). The detected variants were further evaluated by the Polyphen and SIFT software to determine their associations with the pathogenicity of HME.