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.

Haplotype-based approach for noninvasive prenatal tests of Duchenne muscular dystrophy using cell-free fetal DNA in maternal plasma.

PURPOSE: This study demonstrates noninvasive prenatal testing (NIPT) for Duchenne muscular dystrophy (DMD) using a newly developed haplotype-based approach. METHODS: Eight families at risk for DMD were recruited for this study. Parental haplotypes were constructed using target-region sequencing data from the parents and the probands. Fetal haplotypes were constructed using a hidden Markov model through maternal plasma DNA sequencing. The presence of haplotypes linked to the maternal mutant alleles in males indicated affected fetuses. This method was further validated by comparing the inferred single-nucleotide polymorphism (SNP) genotypes to the direct sequencing results of fetal genomic DNA. Prenatal diagnosis was confirmed with amniocentesis, and those results were interpreted in a blinded fashion. RESULTS: The results showed an average accuracy of 99.98% for the total inferred maternal SNPs. With a mean depth of 30× achieved in the 10-Mb target region of each sample, the noninvasive results were consistent with those of the invasive procedure. CONCLUSION: This is the first report of NIPT for DMD and the first application of a haplotype-based approach in NIPT for X-linked diseases. With further improvements in accuracy, this haplotype-based strategy could be feasible for NIPT for DMD and even other X-linked single-gene disorders.

Embryo genome profiling by single-cell sequencing for preimplantation genetic diagnosis in a ß-thalassemia family.

BACKGROUND: The embryonic genome, including genotypes and haplotypes, contains all the information for preimplantation genetic diagnosis, representing great potential for mendelian disorder carriers to conceive healthy babies. METHODS: We developed a strategy to obtain the full embryonic genome for a ß-thalassemia-carrier couple to have a healthy second baby. We carried out sequencing for single blastomere cells and the family trio and further developed the analysis pipeline, including recovery of the missing alleles, removal of the majority of errors, and phasing of the embryonic genome. RESULTS: The final accuracy for homozygous and heterozygous single-nucleotide polymorphisms reached 99.62% and 98.39%, respectively. The aneuploidies of embryos were detected as well. Based on the comprehensive embryonic genome, we effectively performed whole-genome mendelian disorder diagnosis and human leukocyte antigen matching tests. CONCLUSIONS: This retrospective study in a ß-thalassemia family demonstrates a method for embryo genome recovery through single-cell sequencing, which permits detection of genetic variations in preimplantation genetic diagnosis. It shows the potential of single-cell sequencing technology in preimplantation genetic diagnosis clinical practices.

Noninvasive prenatal testing for autosomal recessive conditions by maternal plasma sequencing in a case of congenital deafness.

PURPOSE: The goals of our study were to develop a noninvasive prenatal test for autosomal recessive monogenic conditions and to prove its overall feasibility and potential for clinical integration. METHODS: We recruited a pregnant woman and her spouse, who had a proband child suffering from congenital deafness, and obtained the target-region sequencing data from a semicustom array that used genomic and maternal plasma DNA from three generations of this family. A haplotype-assisted strategy was developed to detect whether the fetus inherited the pathogenic mutations in the causative gene, GJB2. The parental haplotype was constructed using a trio strategy through two different processes, namely, the grandparent-assisted haplotype phasing process and the proband-assisted haplotype phasing process. The fetal haplotype was deduced afterward based on both the maternal plasma sequencing data and the parental haplotype. RESULTS: The accuracy levels of paternal and maternal haplotypes obtained by grandparent-assisted haplotype phasing were 99.01 and 97.36%, respectively, and the proband-assisted haplotype phasing process yielded slightly lower accuracies of 98.73 and 96.79%, respectively. Fetal inheritance of the pathogenic gene was deduced correctly in both processes. CONCLUSION: Our study indicates that the strategy of haplotype-based noninvasive prenatal testing for monogenic conditions has potential applications in clinical practice.

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

OBJECTIVE: 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. METHOD: A double-blind study was performed over 12 months. A total of 189 pregnant women carrying twins were recruited from seven hospitals. Maternal plasma DNA sequencing was performed to detect trisomies 21 and 18. The fetal karyotype was used as gold standard to estimate the sensitivity and specificity of sequencing-based noninvasive prenatal test. RESULTS: There were nine cases of trisomy 21 and two cases of trisomy 18 confirmed by karyotyping. Plasma DNA sequencing correctly identified nine cases of trisomy 21 and one case of trisomy 18. The discordant case of trisomy 18 was an unusual case of monozygotic twin with discordant fetal karyotype (one normal and the other trisomy 18). The sensitivity and specificity of maternal plasma DNA sequencing for fetal trisomy 21 were both 100% and for fetal trisomy 18 were 50% and 100%, respectively. CONCLUSION: Our study further supported that sequencing-based noninvasive prenatal testing of trisomy 21 in twin pregnancies could be achieved with a high accuracy, which could effectively avoid almost 95% of invasive prenatal diagnosis procedures.

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.

Massively parallel sequencing for chromosomal abnormality testing in trophectoderm cells of human blastocysts.

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.

Rapid detection of aneuploidies on a benchtop sequencing platform.

OBJECTIVE: To report a novel method of rapidly detecting fetal aneuploidies for spontaneous abortion using ultra-low whole genome sequencing data on a benchtop sequencing platform. METHOD: Fetal chorionic villus samples were collected from 40 cases of spontaneous abortion with 22 different types of aneuploidy. Genomic DNA of each sample was extracted and sequenced on Illumina MiSeq platform. Unique reads of different read lengths were generated and analyzed using a z-score test. RESULTS: The entire test was finished in 48 hours. An average of 102 k unique reads was obtained for each sample, and all 40 different aneuploidy samples were correctly identified with a z-score of ≥3 or ≤ -3. No false positives or false negatives were observed. Further analysis demonstrated that read length and sequencing type (Paired-end or Single-end) significantly affects the efficiency of sex chromosomal aneuploidy detection. Paired-end 50 bp reads displayed the highest mapping rate and is recommended for future large-scale clinical settings. CONCLUSION: Ultra-low whole genome sequencing can rapidly detect aneuploidy of chromosomes in spontaneous abortion samples in less than 48 hours and therefore can serve as an alternative option to current aneuploidy detection methods for aborted tissues.

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

OBJECTIVE: To report the feasibility of fetal chromosomal deletion/duplication detection using a novel bioinformatic method of low coverage whole genome sequencing of maternal plasma. METHOD: A practical method Fetal Copy-number Analysis through Maternal Plasma Sequencing (FCAPS), integrated with GC-bias correction, binary segmentation algorithm and dynamic threshold strategy, was developed to detect fetal chromosomal deletions/duplications of >10 Mb by low coverage whole genome sequencing (about 0.08-fold). The sensitivity/specificity of the resultant FCAPS algorithm in detecting deletions/duplications was firstly assessed in silico and then tested in 1311 maternal plasma samples from those with known G-banding karyotyping results of the fetus. RESULTS: Deletions/duplications, ranged from 9.01 to 28.46 Mb, were suspected in four of the 1311 samples, of which three were consistent with the results of fetal karyotyping. In one case, the suspected abnormality was not confirmed by karyotyping, representing a false positive case. No false negative case was observed in the remaining 1307 low-risk samples. The sensitivity and specificity for detection of >10-Mb chromosomal deletions/duplications were100% and 99.92%, respectively. CONCLUSION: Our study demonstrated FCAPS has the potential to detect fetal large deletions/duplications (>10 Mb) with low coverage maternal plasma DNA sequencing currently used for fetal aneuploidy detection.

Electrochemical milling and faceting: size reduction and catalytic activation of palladium nanoparticles.

Improved performance through milling: A method for enhancing the catalytic activity of supported metal nanoparticles is reported. This method enhances the activity for the ethanol electro-oxidation of a supported palladium catalyst. The much higher catalytic performance is ascribed to the increased electrochemically active surface area as well as the generation of high-index facets at the milled nanoparticle surface.

Tuning the shape and catalytic activity of Fe nanocrystals from rhombic dodecahedra and tetragonal bipyramids to cubes by electrochemistry.

Fe nanocrystal catalysts were synthesized by electrochemistry. Shape transformations of Fe nanocrystals from rhombic dodecahedra and tetragonal bipyramids, both bounded by {110} facets, to 18-facet polyhedra enclosed by different combinations of {110} and {100} facets and finally to cubes exclusively covered by {100} facets have been achieved. A study of the surface-structure functionality of the Fe nanocrystals toward electroreduction of nitrite revealed that the electrocatalytic activity of the Fe nanocrystals increases as the fraction of {100} facets on the surface of the Fe NCs increases.

[Investigation of electrocatalytic reduction of oxalic acid on Pb electrode through in situ FTIR reflection spectroscopy].

Electrochemical in situ Fourier transform infrared reflection spectroscopy was used in the investigation of electrocatalytic reduction of oxalic acid on Pb electrode. The multi-step potential FTIRS and time-resolved FTIRS procedures were used in the present study. The results of MSFTIRS demonstrate that glyoxylic acid could be detected below -0.70 V. The quantity of glyoxylic acid cumulated on Pb electrode surface reaches a maximum at -0.85 V, then it decreases as electrode potential is further decreased. Meanwhile the C-O stretching vibration of -CH2OH group at around 1 093 cm(-1) could be detected at -0.95 V. It was revealed that all the produced glyoxylic acid may be reduced further into glycolic acid at potentials below -1.50 V. Furthermore, none of other new substances could be detected at more negative potentials, which indicated that glycolic acid could not be further reduced. The results of time resolved Fourier transform infrared reflection spectroscopy at -0.75 V indicate that the integrated intensity of the IR band at about 1 750 cm(-1) for the stretching vibration of C=O (-CHO) linearly increases with the reaction time. The TRFTIR spectra at -1.60 V show that not only the IR absorption of C=O (HOOC-CHO) stretching is observed, but also that of C-O (-CH2OH) stretching at about 1 093 cm(-1) can be seen. The current study demonstrated that electrochemical in situ Fourier transform infrared reflection spectroscopy is a powerful tool for the study of electrosynthesis processes, and for the detection of each species involved in the reaction at molecular level. The results are of significance to understand the reaction mechanism of electrocatalytic reduction of oxalic acid.

[Ethanol electrooxidation on carbon supported PtSn catalyst: in situ TRFTIR study].

Carbon supported PtSn catalyst (PtSn/C) was prepared by a modified polyol method and characterized by means of XRD. It was showed that the metal particle size was 2.2 nm and the unit cell parameter increased compared with Pt/C. In situ time-resolved Fourier transform infrared spectroscopy (TRFTIRS) was used to study the electrooxidation of ethanol on PtSn/C catalyst. COL was the main poison species adsorbed on the active sites to inhibit the further reaction of ethanol electrooxidation. Acetaldehyde and acetic acid were found to be the products of ethanol electrooxidation as competing reactions with ethanol dissociation when the potential was up to 0.3 V, which reduced the poisoning effect. The selectivity of acetic acid among the products was improved with the increase in the potential and reaction time. CO2, which appeared at 0.4 V, was the final product and yielded from the oxidation of COL. The catalytic mechanism of PtSn/C towards ethanol electrooxidation was analyzed based on the results.

[EQCM and in situ FTIR studies on the adsorption and oxidation of 1-butanol at a platinum electrode in alkaline media].

The adsorption and oxidation of 1-butanol in alkaline media on a platinum electrode were investigated mainly by EQCM and in situ FTIR spectroscopy. The experimental results demonstrate that the electrooxidation of 1-butanol is closely relative to solution acidity. Since no chemically adsorbed species, such as CO, were evidenced by in situ FTIR spectroscopy, the adsorption of 1-butanol or its dissociative products on Pt surface is suggested by EQCM and CV data. Only one current peak of 1-butanol oxidation in PGPS was detected at -0.23 V/SCE, which illustrated the disappearance of the second current peak due to Pt electrode passivation in alkaline media. The final product of 1-butanol oxidation is only butyric acid anion under experimental condition. It may therefore be suggested that the main reaction occurring at the electrode is the oxidation of 1-butanol to butyric acid anion. The EQCM studies provide quantitative results of surface mass variation and have shed light on elucidating 1-butanol oxidation.

A new method of ion chromatography technology for speedy determination and analysis in organic electrosynthesis of glyoxylic acid.

Based on ion chromatography (IC) technology, we have developed a new method that combines ion chromatography with a conductivity detector to separate and determine the substances of glyoxal, glycolic acid, oxalic acid and glyoxylic acid. The ion chromatography was applied for the first time in quantitative determination of substances involved in electrosynthesis of glyoxylic acid. The method has been applied to separate and analyze simultaneously either glyoxylic acid and glyoxal in electroxidation of glyoxal, or glyoxylic acid and oxalic acid in electroreduction of oxalic acid. An aqueous Na2CO3-NaHCO3 or NaOH-Na2CO3 solution was confirmed to be the most desirable eluent. The experimental results demonstrated that the detection sensitivity is ahead of ppm grade, and the variation coefficients such as the retention time, the peak height and the peak area outperform 2%. All the recoveries of the detected substances are ranged between 97 and 103%. The method exhibits advantages of high selectivity, high sensitivity, speediness and simple apparatus requirement. Furthermore, simultaneous determination of a mixture of several substances can be achieved by the developed method, and even a neutral molecule of glyoxal can be also determined by choosing an appropriate composition and concentration of eluent.

[In situ FTIR spectroscopy studies of HCOOH oxidation on surface alloy electrocatalysts].

Electrocatalytic properties of three electrodes for formic acid oxidation were studied by using electrochemical in situ FTIR spectroscopy and cyclic voltammetry in this paper. It is demonstrated that the electrocatalytical mechanism of formic acid oxidation on platinum-dispersed carbon(Pt/GC) is similar to that on massive platinum, which involves two paths, i.e. one way through active intermediate and the other through poison intermediate to CO2. The Pt/GC exhibits higher catalytivity than pure platinum. The electrode of Pt/GC modified by Sb (Sb-Pt/GC) was also prepared in the work. It was observed that the onset potential (Ei) for formic acid oxidation on Sb-Pt/GC was shift negatively for 0.20 V. The peak potential (Ep) was observed to shifted negatively to 0.34 V and the value of oxidation current (jp) was enhanced nearly 7.28 times. Similar results were also observed on surface alloy/GC prepared. In this case, Ei and Ep were -0.12 and 0.32 V, respectively, jp was enhanced about 8.15 times, and FWHM (full width at half maximum) was 0.50 V. It is indicated that Sb-Pt/GC and surface alloy/GC can not only effectively restrain the formation of poison intermediate CO, but also significantly increase the electrocatalytic activities for oxidation of active intermediates.

[Solid/liquid interfacial in situ IR microscope and step-scan time-resolved FTIR spectroscopy and applications in studies of nanomaterials].

Electrochemical in situ microscope IR reflection spectroscopy and step-scan time-resolved FTIR reflection spectroscopy were established by using an IR-plan advantage microscope and a Nexus 870 FTIR instrument, and a home-made signal synchronizer that harmonizes electrode polarization potential and step-scan spectral data collection sequence. These new techniques have been applied in studies of particular IR properties of 2-dimensional nanomaterials. By applying a treatment of fast potential cycling with different time (tau), a set of nanostructured Pt microelectrodes were prepared. CO adsorption was employed as a probe reaction together with in situ developed microscope FTIR spectroscopy. The results illustrated the variation of abnormal IR features with the nanostructure and the thickness (i.e., the size) of film formed on Pt microelectrode, i.e., following the increase of tau in fast potential cycling treatment, the direction of CO band was turned from absorption to antiabsorption direction, and the intensity and the width of CO band were increased. By taking the advantage of the abnormal infrared effects of nanostructured Pt microelectrode, the sensitivity of in situ IR reflection spectroscopy has been significantly improved, and spectra of time-resolution as fast as 50 micros have been recorded at solid/liquid interfaces. The current studies demonstrated not only the success of development of new techniques of in situ IR spectroscopy, but also the exploitation of the established techniques in studies of nanomaterials.

Non-invasive fetal sex determination by maternal plasma sequencing and application in X-linked disorder counseling.

OBJECTIVE: To develop a fetal sex determination method based on maternal plasma sequencing (MPS), assess its performance and potential use in X-linked disorder counseling. METHODS: 900 cases of MPS data from a previous study were reviewed, in which 100 and 800 cases were used as training and validation set, respectively. The percentage of uniquely mapped sequencing reads on Y chromosome was calculated and used to classify male and female cases. Eight pregnant women who are carriers of Duchenne muscular dystrophy (DMD) mutations were recruited, whose plasma were subjected to multiplex sequencing and fetal sex determination analysis. RESULTS: In the training set, a sensitivity of 96% and false positive rate of 0% for male cases detection were reached in our method. The blinded validation results showed 421 in 423 male cases and 374 in 377 female cases were successfully identified, revealing sensitivity and specificity of 99.53% and 99.20% for fetal sex determination, at as early as 12 gestational weeks. Fetal sex for all eight DMD genetic counseling cases were correctly identified, which were confirmed by amniocentesis. CONCLUSIONS: Based on MPS, high accuracy of non-invasive fetal sex determination can be achieved. This method can potentially be used for prenatal genetic counseling.

Performance comparison between rapid sequencing platforms for ultra-low coverage sequencing strategy.

Ultra-low coverage sequencing (ULCS) is one of the most promising strategies for sequencing based clinical application. These clinical applications, especially prenatal diagnosis, have a strict requirement of turn-around-time; therefore, the application of ULCS is restricted by current high throughput sequencing platforms. Recently, the emergence of rapid sequencing platforms, such as MiSeq and Ion Proton, brings ULCS strategy into a new era. The comparison of their performance could shed lights on their potential application in large-scale clinic trials. In this study, we performed ULCS (<0.1X coverage) on both MiSeq and Ion Proton platforms for 18 spontaneous abortion fetuses carrying aneuploidy and compared their performance on different levels. Overall basic data and GC bias showed no significant difference between these two platforms. We also found the sex and aneuploidy detection indicated 100% sensitivity and 100% specificity on both platforms. Our study generated essential data from these two rapid sequencing platforms, which provides useful reference for later research and potentially accelerates the clinical applications of ULCS.

PSCC: sensitive and reliable population-scale copy number variation detection method based on low coverage sequencing.

BACKGROUND: Copy number variations (CNVs) represent an important type of genetic variation that deeply impact phenotypic polymorphisms and human diseases. The advent of high-throughput sequencing technologies provides an opportunity to revolutionize the discovery of CNVs and to explore their relationship with diseases. However, most of the existing methods depend on sequencing depth and show instability with low sequence coverage. In this study, using low coverage whole-genome sequencing (LCS) we have developed an effective population-scale CNV calling (PSCC) method. METHODOLOGY/PRINCIPAL FINDINGS: In our novel method, two-step correction was used to remove biases caused by local GC content and complex genomic characteristics. We chose a binary segmentation method to locate CNV segments and designed combined statistics tests to ensure the stable performance of the false positive control. The simulation data showed that our PSCC method could achieve 99.7%/100% and 98.6%/100% sensitivity and specificity for over 300 kb CNV calling in the condition of LCS (∼2×) and ultra LCS (∼0.2×), respectively. Finally, we applied this novel method to analyze 34 clinical samples with an average of 2× LCS. In the final results, all the 31 pathogenic CNVs identified by aCGH were successfully detected. In addition, the performance comparison revealed that our method had significant advantages over existing methods using ultra LCS. CONCLUSIONS/SIGNIFICANCE: Our study showed that PSCC can sensitively and reliably detect CNVs using low coverage or even ultra-low coverage data through population-scale sequencing.