Ph.D. in Ophthalmology, Jinan University, 2008
M.S. in Otolaryngology, Jinan University, 2005
M.D. in Clinical Medicine, Jinan University, 2002
2013-2014: Visiting scholar at the Wang Genomics Lab
Deputy Director of the Physicians, Jinan University Hospital; Associate Professor, Jinan University
Long-range PCR in next-generation sequencing, Exome sequencing on meningiomas
Long-range PCR in next-generation sequencing
Background: Long-range PCR remains a flexible, fast, efficient and cost-effective choice for sequencing candidate genomic regions in a small number of samples, especially when combined with next-generation sequencing (NGS) platforms. Several long-range DNA polymerases are advertised as being able to amplify up to 15kb or longer genomic DNA. However, their real-world performance characteristics and their suitability for NGS remain unclear.
Methods: We evaluated six long-range DNA polymerases to amplify three amplicons, with sizes of 12.9kb, 9.7kb, and 5.8kb, respectively. These enzymes include Invitrogen SequalPrep, Invitrogen AccuPrime, TaKaRa PrimeSTAR GXL, TaKaRa LA Taq Hot Start, KAPA Long Range HotStart and QIAGEN LongRange PCR Polymerase. Subsequently, we used the PrimeSTAR enzyme to amplify entire BRCA1 (83.2kb) and BRCA2 (84.2kb) genes from nine subjects, generated libraries with the Nextera XT library preparation protocol, and sequenced them on an Illumina MiSeq sequencer.
Results: We found that the TaKaRa PrimeSTAR GXL DNA polymerase can amplify almost all amplicons with different sizes and Tm values under identical PCR conditions. Other enzymes require alteration of PCR conditions to obtain optimal performance. From the MiSeq run, we identified multiple intronic and exonic single-nucleotide variations (SNVs), including one mutation (c.5946delT in BRCA2) in a positive control.
Conclusions: Our results provided a useful guide on how to use long-range PCR to perform NGS on large genomic regions, especially when the entire gene regions including introns are of interest.
Exome sequencing on meningiomas
BACKGROUND: Meningiomas are tumors originating from the membranous layers surrounding the central nervous system, and are generally regarded as “benign” tumors of the brain. Although extremely rare, the malignant meningiomas are typically associated with a higher risk of local tumor recurrence and a poorer prognosis (median survival time <2 years). Previous genome-wide association studies and exome sequencing studies have identified genes that play a role in susceptibility or progression in meningiomas, but these studies did not focus specifically on malignant tumors.
METHODS: We performed exome sequencing on five malignant meningiomas on the Illumina HiSeq2000 platform using Agilent SureSelect Human All Exon Kits, identified candidate genes with recurrent mutations, and validated selected mutations by Sanger sequencing. We next designed custom NimbleGen targeted region arrays on five candidate genes, and sequenced four additional malignant meningiomas. We also used the MutSigCV software and a novel algorithm built on MetaLR scores to generate additional lists of potential cancer driver genes for further replication studies.
RESULTS: From exome sequencing data, we identified several frequently mutated genes including NF2, MN1, ARID1B, SEMA4D and MUC2, with private mutations in tumors. We sequenced these genes in four additional samples and identified potential driver mutations in NF2, MN1 and MUC2. A large number of mutations MUC2 were found, which may be due to alignment and variant calling artifacts. Additional exploratory analysis on exome data identified genes enriched in notch-signaling pathway that may lead to malignancy.
CONCLUSIONS: We confirmed that mutations in NF2 are likely to play a role in progression of meningiomas, and nominated MN1 as a candidate gene for malignant transformation of meningiomas. Our sample size is limited by the extreme rarity of malignant meningiomas, but our study provided a prioritized list of genes to be replicated in future studies with additional samples.
- Zhang X*, Jia H*, Lu Y*, Dong C, Hou J, Wang Z, Wang F, Zhong H, Wang L, Wang K. Exome sequencing on malignant meningiomas identified mutations in NF2 and MN1. Discovery Medicine, in press, 2014
- Jia H, Guo Y, Zhao W, Wang K. Long-range PCR in next-generation sequencing: comparison of six enzymes and evaluation on the MiSeq sequencer. Scientific Reports, 4:5737, 2014