Performance characterization of PCR-free whole genome sequencing for clinical diagnosis

Guiju Zhou, Meizhen Zhou, Fanwei Zeng, Ningzhi Zhang, Yan Sun, Zhihong Qiao, Xueqin Guo, Shihao Zhou, Guojun Yun, Jiansheng Xie, Xiaodan Wang, Fengxia Liu, Chunna Fan, Yaoshen Wang, Zhonghai Fang, Zhongming Tian, Wentao Dai, Jun Sun, Zhiyu Peng, Lijie Song*

*Corresponding author for this work

Research output: Contribution to journalJournal articleResearchpeer-review

136 Downloads (Pure)


To evaluate the performance of polymerase chain reaction (PCR)-free whole genome sequencing (WGS) for clinical diagnosis, and thereby revealing how experimental parameters affect variant detection.Five NA12878 samples were sequenced using MGISEQ-2000. NA12878 samples underwent WGS with differing deoxyribonucleic acid (DNA) input and library preparation protocol (PCR-based vs PCR-free protocols for library preparation). The depth of coverage and genotype quality of each sample were compared. The performance of each sample was measured for sensitivity, coverage of depth and breadth of coverage of disease-related genes, and copy number variants. We also developed a systematic WGS pipeline (PCR-free) for the analysis of 11 clinical cases.In general, NA12878-2 (PCR-free WGS) showed better depth of coverage and genotype quality distribution than NA12878-1 (PCR-based WGS). With a mean depth of ∼40×, the sensitivity of homozygous and heterozygous single nucleotide polymorphisms (SNPs) of NA12878-2 showed higher sensitivity (>99.77% and >99.82%) than NA12878-1, and positive predictive value exceeded 99.98% and 99.07%. The sensitivity and positive predictive value of homozygous and heterozygous indels for NA12878-2 (PCR-free WGS) showed great improvement than NA128878-1. The breadths of coverage for disease-related genes and copy number variants are slightly better for samples with PCR-free library preparation protocol than the sample with PCR-based library preparation protocol. DNA input also influences the performance of variant detection in samples with PCR-free WGS. All the 19 previously confirmed variants in 11 clinical cases were successfully detected by our WGS pipeline (PCR free).Different experimental parameters may affect variant detection for clinical WGS. Clinical scientists should know the range of sensitivity of variants for different methods of WGS, which would be useful when interpreting and delivering clinical reports.
Original languageEnglish
Article numbere28972
JournalMedicine (United States)
Issue number10
Publication statusPublished - 2022


  • Clinical diagnosis
  • Deoxyribonucleic acid input
  • Polymerase chain reaction-free
  • Sequencing depth and coverage
  • Whole genome sequencing


Dive into the research topics of 'Performance characterization of PCR-free whole genome sequencing for clinical diagnosis'. Together they form a unique fingerprint.

Cite this