Shengpei Chen

Whole genome sequencing identifies recurrent mutations in hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is one of the most deadly cancers worldwide and has no effective treatment, yet the molecular basis of hepatocarcinogenesis remains largely unknown. Here we report findings from a whole-genome sequencing (WGS) study of 88 matched HCC tumor/normal pairs, 81 of which are Hepatitis B virus (HBV) positive, seeking to identify genetically altered genes and pathways implicated in HBV-associated HCC. We find beta-catenin to be the most frequently mutated oncogene (15.9%) and TP53 the most frequently mutated tumor suppressor (35.2%). The Wnt/beta-catenin and JAK/STAT pathways, altered in 62.5% and 45.5% of cases, respectively, are likely to act as two major oncogenic drivers in HCC. This study also identifies several prevalent and potentially actionable mutations, including activating mutations of Janus kinase 1 (JAK1), in 9.1% of patients and provides a path toward therapeutic intervention of the disease.

Noninvasive prenatal diagnosis of common fetal chromosomal aneuploidies by maternal plasma DNA sequencing

Objective: To develop a new bioinformatic method in the noninvasive prenatal identification of common fetal aneuploidies using massively parallel sequencing on maternal plasma. Methods: Massively parallel sequencing was performed on plasma DNA samples from 108 pregnant women (median gestation: 12+5 week) immediately before chorionic villus sampling (CVS) or amniocentesis. Data were analysed using a novel z-score method with internal reference chromosome. The diagnostic accuracies of the fetal karyotyping status were compared against two previously reported z-score methods – one without adjustment and the other with GC correction. Results: A total of 32 cases with fetal aneuploidy were confirmed by conventional karyotyping, including 11 cases of Trisomy 21, 10 cases of Trisomy 18, 2 cases of Trisomy 13, 8 cases of Turner syndrome (45, XO) and one case of Klinefelter syndrome (47, XXY). Using the z-score method without reference adjustment, the detection rate for Trisomy 21, Trisomy 18, Trisomy 13, Turner syndrome, and Klinefelter’s syndrome is 100%, 40%, 0%, 88% and 0% respectively. Using the z-score method with GC correction, the detection rate increased to 100% for Trisomy 21, 90% for Trisomy 18, 100% for Trisomy 13. By using the z-score method with internal reference, the detection rate increased to 100% for all aneuploidies. The false positive rate was 0% for all three methods. Conclusion: This massively parallel sequencing-based approach, combined with the improved z-score test methodology, enables the prenatal diagnosis of most common aneuploidies with a high degree of accuracy, even in the first trimester of pregnancy.

Noninvasive Fetal Trisomy (NIFTY) test: an advanced noninvasive prenatal diagnosis methodology for fetal autosomal and sex chromosomal aneuploidies

BackgroundConventional prenatal screening tests, such as maternal serum tests and ultrasound scan, have limited resolution and accuracy.MethodsWe developed an advanced noninvasive prenatal diagnosis method based on massively parallel sequencing. The Noninvasive Fetal Trisomy (NIFTY) test, combines an optimized Student’s t-test with a locally weighted polynomial regression and binary hypotheses. We applied the NIFTY test to 903 pregnancies and compared the diagnostic results with those of full karyotyping.Results16 of 16 trisomy 21, 12 of 12 trisomy 18, two of two trisomy 13, three of four 45, X, one of one XYY and two of two XXY abnormalities were correctly identified. But one false positive case of trisomy 18 and one false negative case of 45, X were observed. The test performed with 100% sensitivity and 99.9% specificity for autosomal aneuploidies and 85.7% sensitivity and 99.9% specificity for sex chromosomal aneuploidies. Compared with three previously reported z-score approaches with/without GC-bias removal and with internal control, the NIFTY test was more accurate and robust for the detection of both autosomal and sex chromosomal aneuploidies in fetuses.ConclusionOur study demonstrates a powerful and reliable methodology for noninvasive prenatal diagnosis.

Massively Parallel Sequencing for Chromosomal Abnormality Testing in Trophectoderm Cells of Human Blastocysts

ABSTRACT Preimplantation genetic diagnosis and screening are widely accepted for chromosomal abnormality identification to avoid transferring embryos with genetic defects. Massively parallel sequencing (MPS) is a rapidly developing approach for genome analysis with increasing application in clinical practice. The purpose of this study was to use MPS for identification of aneuploidies and unbalanced chromosomal rearrangements after blastocyst biopsy. Trophectoderm (TE) samples of 38 blastocysts from 16 in vitro fertilization cycles were subjected to analysis. Low-coverage whole genome sequencing was performed using the Illumina HiSeq2000 platform with a novel algorithm purposely created for chromosomal analysis. The efficiency of this MPS approach was estimated by comparing results obtained by an Affymetrix single-nucleotide polymorphism (SNP) array. Whole genome amplification (WGA) products of TE cells were detected by MPS, with an average of 0.07× depth and 5.5% coverage of the human genome. Twenty-six embryos (68.4%) were detected as euploid, while six embryos (15.8%) contained uniform aneuploidies. Four of these (10.5%) were with solely unbalanced chromosomal rearrangements, whereas the remaining two embryos (5.3%) showed both aneuploidies and unbalanced rearrangements. Almost all these results were confirmed by the SNP array, with the exception of one sample, where different sizes of unbalanced rearrangements were detected, possibly due to chromosomal GC bias in array analysis. Our study demonstrated MPS could be applied to accurately detect embryonic chromosomal abnormality with a flexible and cost-effective strategy and higher potential accuracy.

A method for noninvasive detection of fetal large deletions/ duplications by low coverage massively parallel sequencing

To report the feasibility of fetal chromosomal deletion/duplication detection using a novel bioinformatic method of low coverage whole genome sequencing of maternal plasma.

A single cell level based method for copy number variation analysis by low coverage massively parallel sequencing.

Copy number variations (CNVs), a common genomic mutation associated with various diseases, are important in research and clinical applications. Whole genome amplification (WGA) and massively parallel sequencing have been applied to single cell CNVs analysis, which provides new insight for the fields of biology and medicine. However, the WGA-induced bias significantly limits sensitivity and specificity for CNVs detection. Addressing these limitations, we developed a practical bioinformatic methodology for CNVs detection at the single cell level using low coverage massively parallel sequencing. This method consists of GC correction for WGA-induced bias removal, binary segmentation algorithm for locating CNVs breakpoints, and dynamic threshold determination for final signals filtering. Afterwards, we evaluated our method with seven test samples using low coverage sequencing (4∼9.5%). Four single-cell samples from peripheral blood, whose karyotypes were confirmed by whole genome sequencing analysis, were acquired. Three other test samples derived from blastocysts whose karyotypes were confirmed by SNP-array analysis were also recruited. The detection results for CNVs of larger than 1 Mb were highly consistent with confirmed results reaching 99.63% sensitivity and 97.71% specificity at base-pair level. Our study demonstrates the potential to overcome WGA-bias and to detect CNVs (>1 Mb) at the single cell level through low coverage massively parallel sequencing. It highlights the potential for CNVs research on single cells or limited DNA samples and may prove as a promising tool for research and clinical applications, such as pre-implantation genetic diagnosis/screening, fetal nucleated red blood cells research and cancer heterogeneity analysis.

Clinical outcome of preimplantation genetic diagnosis and screening using next generation sequencing

BackgroundNext generation sequencing (NGS) is now being used for detecting chromosomal abnormalities in blastocyst trophectoderm (TE) cells from in vitro fertilized embryos. However, few data are available regarding the clinical outcome, which provides vital reference for further application of the methodology. Here, we present a clinical evaluation of NGS-based preimplantation genetic diagnosis/screening (PGD/PGS) compared with single nucleotide polymorphism (SNP) array-based PGD/PGS as a control.ResultsA total of 395 couples participated. They were carriers of either translocation or inversion mutations, or were patients with recurrent miscarriage and/or advanced maternal age. A total of 1,512 blastocysts were biopsied on D5 after fertilization, with 1,058 blastocysts set aside for SNP array testing and 454 blastocysts for NGS testing. In the NGS cycles group, the implantation, clinical pregnancy and miscarriage rates were 52.6% (60/114), 61.3% (49/80) and 14.3% (7/49), respectively. In the SNP array cycles group, the implantation, clinical pregnancy and miscarriage rates were 47.6% (139/292), 56.7% (115/203) and 14.8% (17/115), respectively. The outcome measures of both the NGS and SNP array cycles were the same with insignificant differences. There were 150 blastocysts that underwent both NGS and SNP array analysis, of which seven blastocysts were found with inconsistent signals. All other signals obtained from NGS analysis were confirmed to be accurate by validation with qPCR. The relative copy number of mitochondrial DNA (mtDNA) for each blastocyst that underwent NGS testing was evaluated, and a significant difference was found between the copy number of mtDNA for the euploid and the chromosomally abnormal blastocysts. So far, out of 42 ongoing pregnancies, 24 babies were born in NGS cycles; all of these babies are healthy and free of any developmental problems.ConclusionsThis study provides the first evaluation of the clinical outcomes of NGS-based pre-implantation genetic diagnosis/screening, and shows the reliability of this method in a clinical and array-based laboratory setting. NGS provides an accurate approach to detect embryonic imbalanced segmental rearrangements, to avoid the potential risks of false signals from SNP array in this study.

Noninvasive prenatal testing of trisomies 21 and 18 by massively parallel sequencing of maternal plasma DNA in twin pregnancies.

The objective of this study is to assess the performance of noninvasive prenatal testing for trisomies 21 and 18 on the basis of massively parallel sequencing of cell‐free DNA from maternal plasma in twin pregnancies.

Haplotype-assisted accurate non-invasive fetal whole genome recovery through maternal plasma sequencing

BackgroundThe applications of massively parallel sequencing technology to fetal cell-free DNA (cff-DNA) have brought new insight to non-invasive prenatal diagnosis. However, most previous research based on maternal plasma sequencing has been restricted to fetal aneuploidies. To detect specific parentally inherited mutations, invasive approaches to obtain fetal DNA are the current standard in the clinic because of the experimental complexity and resource consumption of previously reported non-invasive approaches.MethodsHere, we present a simple and effective non-invasive method for accurate fetal genome recovery-assisted with parental haplotypes. The parental haplotype were firstly inferred using a combination strategy of trio and unrelated individuals. Assisted with the parental haplotype, we then employed a hidden Markov model to non-invasively recover the fetal genome through maternal plasma sequencing.ResultsUsing a sequence depth of approximately 44X against a an approximate 5.69% cff-DNA concentration, we non-invasively inferred fetal genotype and haplotype under different situations of parental heterozygosity. Our data show that 98.57%, 95.37%, and 98.45% of paternal autosome alleles, maternal autosome alleles, and maternal chromosome X in the fetal haplotypes, respectively, were recovered accurately. Additionally, we obtained efficient coverage or strong linkage of 96.65% of reported Mendelian-disorder genes and 98.90% of complex disease-associated markers.ConclusionsOur method provides a useful strategy for non-invasive whole fetal genome recovery.

HIVID: An efficient method to detect HBV integration using low coverage sequencing

We reported HIVID (high-throughput Viral Integration Detection), a novel experimental and computational method to detect the location of Hepatitis B Virus (HBV) integration breakpoints in Hepatocellular Carcinoma (HCC) genome. In this method, the fragments with HBV sequence were enriched by a set of HBV probes and then processed to high-throughput sequencing. In order to evaluate the performance of HIVID, we compared the results of HIVID with that of whole genome sequencing method (WGS) in 28 HCC tumors. We detected a total of 246 HBV integration breakpoints in HCC genome, 113 out of which were within 400bp upstream or downstream of 125 breakpoints identified by WGS method, covering 89.3% (125/140) of total breakpoints. The integration was located in the gene TERT, MLL4, and CCNE1. In addition, we discovered 133 novel breakpoints missed by WGS method, with 66.7% (10/15) of validation rate. Our study shows HIVID is a cost-effective methodology with high specificity and sensitivity to identify viral integration in human genome.