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.