New insights into the performance of human whole-exome capture platforms

Whole exome sequencing (WES) is increasingly used in research and diagnostics. WES users expect coverage of the entire coding region of known genes as well as sufficient read depth for the covered regions. It is, however, unknown which recent WES platform is most suitable to meet these expectations. We present insights into the performance of the most recent standard exome enrichment platforms from Agilent, NimbleGen and Illumina applied to six different DNA samples by two sequencing vendors per platform. Our results suggest that both Agilent and NimbleGen overall perform better than Illumina and that the high enrichment performance of Agilent is stable among samples and between vendors, whereas NimbleGen is only able to achieve vendor- and sample-specific best exome coverage. Moreover, the recent Agilent platform overall captures more coding exons with sufficient read depth than NimbleGen and Illumina. Due to considerable gaps in effective exome coverage, however, the three platforms cannot capture all known coding exons alone or in combination, requiring improvement. Our data emphasize the importance of evaluation of updated platform versions and suggest that enrichment-free whole genome sequencing can overcome the limitations of WES in sufficiently covering coding exons, especially GC-rich regions, and in characterizing structural variants.     

Multiple-laboratory comparison of microarray platforms

Microarray technology is a powerful tool for measuring RNA expression for thousands of genes at once. Various studies have been published comparing competing platforms with mixed results: some find agreement, others do not. As the number of researchers starting to use microarrays and the number of cross-platform meta-analysis studies rapidly increases, appropriate platform assessments become more important. Here we present results from a comparison study that offers important improvements over those previously described in the literature. In particular, we noticed that none of the previously published papers consider differences between labs. For this study, a consortium of ten laboratories from the Washington, DC-Baltimore, USA, area was formed to compare data obtained from three widely used platforms using identical RNA samples. We used appropriate statistical analysis to demonstrate that there are relatively large differences in data obtained in labs using the same platform, but that the results from the best-performing labs agree rather well.     

Performance comparison of whole-genome sequencing platforms

Whole-genome sequencing is becoming commonplace, but the accuracy and completeness of variant calling by the most widely used platforms from Illumina and Complete Genomics have not been reported. Here we sequenced the genome of an individual with both technologies to a high average coverage of ～76×, and compared their performance with respect to sequence coverage and calling of single-nucleotide variants (SNVs), insertions and deletions (indels). Although 88.1% of the ～3.7 million unique SNVs were concordant between platforms, there were tens of thousands of platform-specific calls located in genes and other genomic regions. In contrast, 26.5% of indels were concordant between platforms. Target enrichment validated 92.7% of the concordant SNVs, whereas validation by genotyping array revealed a sensitivity of 99.3%. The validation experiments also suggested that >60% of the platform-specific variants were indeed present in the genome. Our results have important implications for understanding the accuracy and completeness of the genome sequencing platforms.     

Evaluation of gene expression measurements from commercial microarray platforms

Multiple commercial microarrays for measuring genome-wide gene expression levels are currently available, including oligonucleotide and cDNA, single- and two-channel formats. This study reports on the results of gene expression measurements generated from identical RNA preparations that were obtained using three commercially available microarray platforms. RNA was collected from PANC-1 cells grown in serum-rich medium and at 24 h following the removal of serum. Three biological replicates were prepared for each condition, and three experimental replicates were produced for the first biological replicate. RNA was labeled and hybridized to microarrays from three major suppliers according to manufacturers’ protocols, and gene expression measurements were obtained using each platform’s standard software. For each platform, gene targets from a subset of 2009 common genes were compared. Correlations in gene expression levels and comparisons for significant gene expression changes in this subset were calculated, and showed considerable divergence across the different platforms, suggesting the need for establishing industrial manufacturing standards, and further independent and thorough validation of the technology.     

Performance comparison of benchtop high-throughput sequencing platforms

Three benchtop high-throughput sequencing instruments are now available. The 454 GS Junior (Roche), MiSeq (Illumina) and Ion Torrent PGM (Life Technologies) are laser-printer sized and offer modest set-up and running costs. Each instrument can generate data required for a draft bacterial genome sequence in days, making them attractive for identifying and characterizing pathogens in the clinical setting. We compared the performance of these instruments by sequencing an isolate of Escherichia coli O104:H4, which caused an outbreak of food poisoning in Germany in 2011. The MiSeq had the highest throughput per run (1.6 Gb/run, 60 Mb/h) and lowest error rates. The 454 GS Junior generated the longest reads (up to 600 bases) and most contiguous assemblies but had the lowest throughput (70 Mb/run, 9 Mb/h). Run in 100-bp mode, the Ion Torrent PGM had the highest throughput (80–100 Mb/h). Unlike the MiSeq, the Ion Torrent PGM and 454 GS Junior both produced homopolymer-associated indel errors (1.5 and 0.38 errors per 100 bases, respectively).     

Transcriptional signatures of normal human mammary epithelial cells in response to benzo[a]pyrene exposure: a comparison of three microarray platforms

Microarrays are used to study gene expression in a variety of biological systems. A number of different platforms have been developed, but few studies exist that have directly compared the performance of one platform with another. The goal of this study was to determine array variation by analyzing the same RNA samples with three different array platforms. Using gene expression responses to benzo[a]pyrene exposure in normal human mammary epithelial cells (NHMECs), we compared the results of gene expression profiling using three microarray platforms: photolithographic oligonucleotide arrays (Affymetrix), spotted oligonucleotide arrays (Amersham), and spotted cDNA arrays (NCI). While most previous reports comparing microarrays have analyzed pre-existing data from different platforms, this comparison study used the same sample assayed on all three platforms, allowing for analysis of variation from each array platform. In general, poor correlation was found with corresponding measurements from each platform. Each platform yielded different gene expression profiles, suggesting that while microarray analysis is a useful discovery tool, further validation is needed to extrapolate results for broad use of the data. Also, microarray variability needs to be taken into consideration, not only in the data analysis but also in specific probe selection for each array type.     

Comparison of sequence reads obtained from three next-generation sequencing platforms

Next-generation sequencing technologies enable the rapid cost-effective production of sequence data. To evaluate the performance of these sequencing technologies, investigation of the quality of sequence reads obtained from these methods is important. In this study, we analyzed the quality of sequence reads and SNP detection performance using three commercially available next-generation sequencers, i.e., Roche Genome Sequencer FLX System (FLX), Illumina Genome Analyzer (GA), and Applied Biosystems SOLiD system (SOLiD). A common genomic DNA sample obtained from Escherichia coli strain DH1 was applied to these sequencers. The obtained sequence reads were aligned to the complete genome sequence of E. coli DH1, to evaluate the accuracy and sequence bias of these sequence methods. We found that the fraction of "junk" data, which could not be aligned to the reference genome, was largest in the data set of SOLiD, in which about half of reads could not be aligned. Among data sets after alignment to the reference, sequence accuracy was poorest in GA data sets, suggesting relatively low fidelity of the elongation reaction in the GA method. Furthermore, by aligning the sequence reads to the E. coli strain W3110, we screened sequence differences between two E. coli strains using data sets of three different next-generation platforms. The results revealed that the detected sequence differences were similar among these three methods, while the sequence coverage required for the detection was significantly small in the FLX data set. These results provided valuable information on the quality of short sequence reads and the performance of SNP detection in three next-generation sequencing platforms.     

Comparison and evaluation of two exome capture kits and sequencing platforms for variant calling

Background

To promote the clinical application of next-generation sequencing, it is important to obtain accurate and consistent variants of target genomic regions at low cost. Ion Proton, the latest updated semiconductor-based sequencing instrument from Life Technologies, is designed to provide investigators with an inexpensive platform for human whole exome sequencing that achieves a rapid turnaround time. However, few studies have comprehensively compared and evaluated the accuracy of variant calling between Ion Proton and Illumina sequencing platforms such as HiSeq 2000, which is the most popular sequencing platform for the human genome. The Ion Proton sequencer combined with the Ion TargetSeq™ Exome Enrichment Kit together make up TargetSeq-Proton, whereas SureSelect-Hiseq is based on the Agilent SureSelect Human All Exon v4 Kit and the HiSeq 2000 sequencer.

Results

Here, we sequenced exonic DNA from four human blood samples using both TargetSeq-Proton and SureSelect-HiSeq. We then called variants in the exonic regions that overlapped between the two exome capture kits (33.6 Mb). The rates of shared variant loci called by two sequencing platforms were from 68.0 to 75.3 % in four samples, whereas the concordance of co-detected variant loci reached 99 %. Sanger sequencing validation revealed that the validated rate of concordant single nucleotide polymorphisms (SNPs) (91.5 %) was higher than the SNPs specific to TargetSeq-Proton (60.0 %) or specific to SureSelect-HiSeq (88.3 %). With regard to 1-bp small insertions and deletions (InDels), the Sanger sequencing validated rates of concordant variants (100.0 %) and SureSelect-HiSeq-specific (89.6 %) were higher than those of TargetSeq-Proton-specific (15.8 %).

Conclusions

In the sequencing of exonic regions, a combination of using of two sequencing strategies (SureSelect-HiSeq and TargetSeq-Proton) increased the variant calling specificity for concordant variant loci and the sensitivity for variant loci called by any one platform. However, for the sequencing of platform-specific variants, the accuracy of variant calling by HiSeq 2000 was higher than that of Ion Proton, specifically for the InDel detection. Moreover, the variant calling software also influences the detection of SNPs and, specifically, InDels in Ion Proton exome sequencing.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1796-6) contains supplementary material, which is available to authorized users.     

Micropolyspora faeni antigens of three commercial extracts

The protein composition of three commercial extracts of Micropolyspora faeni, produced in U.S.A., England and Italy has been evaluated by agarose gel electrophoresis.By crossed immunoelectrophoresis, tandem-crossed immunoelectrophoresis and by a modification of this last technique, the antigenic composition and the common antigens of the extracts have been investigated. Hyperimmune rabbit serum and a pool of five human sera with precipitins to Micropolyspora faeni have been used as source of antibody.Different quantity and quality of protein content was observed in the available batches. Different antigenic composition was also observed, not directly related to the different proteins contained therein; three antigens were definitely common to all extracts and two of them represented the major antigens of each extract.Despite the total protein content, the major and common antigens were found in similar concentrations in all three products examined. Therefore, the discrepancies observed in the precipitin reactions using the three commercial Micropolyspora faeni extracts are due to differences in the minor antigen composition of the extracts.     

Comparison of the three optical platforms for measurement of cellular respiration

We compared three optical platforms for measurement of cellular respiration: absolute oxygen consumption rates (OCRs) in hermetically sealed microcuvettes, relative OCRs measured in a 96-well plate with oil seal, and steady-state oxygenation of cells in an open 96-well plate. Using mouse embryonic fibroblasts cell line, the phosphorescent intracellular O₂ probe MitoXpress-Intra, and time-resolved fluorescence reader, we determined algorithms for conversion of relative OCRs and cell oxygenation into absolute OCRs, thereby allowing simple high-throughput measurement of absolute OCR values.     

Biomimetic platforms for human stem cell research

Stem cells are central to developing new treatment options for tissue regeneration and constructing controllable models for biological research. Bioengineered cell culture environments that combine microenvironmental control with tissue-specific transport and signaling are critical tools in our efforts to study tissue development, regeneration, and disease under conditions that predict the human in vivo context. We propose that experimentation at the interfaces of biology, engineering, and medical sciences is critical for unlocking the full potential of stem cells. Here, we focus on the design and utilization of in vitro platforms that recapitulate the environments associated with tissue development, disease, and regeneration.     

The present and future of whole-exome sequencing in studying and treating human reproductive disorders

The causes of recurrent spontaneous abortion (RSA) and fetal malformations are multifactorial and unclear in most cases. Environmental, maternal, and genetic factors have been shown to contribute to these defects. Whole-exome sequencing (WES) is widely used to detect genetic variations associated with human diseases and has recently been successfully applied to unveil genetic causes of unexplained recurrent spontaneous abortion (URSA) and fetal malformations. Here, we review the current discovery and diagnosis strategies to identify the underlying pathogenic mutations of URSA and fetal malformations using WES technology and propose to further develop WES, both to advance our understanding of these diseases and to eventually lead to targeted therapies for reproductive disorders.     

Determination of urinary cortisol with three commercial immunoassays.

Urinary cortisol determination was performed with three commercially available immunoassays: one enzyme-immunoassay (Cortisol Biotrol) (EIA) and two radioimmunoassays: Quanticoat Cortisol (Kallestad Diagnostics) (KD-RIA) and GammaCoat Cortisol (Clinical Assays) (CA-RIA). Four procedures were carried out. Procedure I (methylene chloride extraction) was applied to EIA and CA-RIA and procedure II (ethyl acetate extraction) to KD-RIA. Procedure III combining procedure I and column chromatography on Sephadex LH 20 in methylene chloride was applied to the three kits. Procedure IV consisting of carbon tetrachloride preextraction and extraction with cyclohexane-ethyl acetate (50:50, v/v) was applied to CA-RIA. The results obtained were compared with those of the reference technique, "on-line" HPLC with u.v. detection. Two groups of results were arbitrarily considered, those below (n = 28) and those above (n = 6) 270 nmol/l. In the first group, the results were markedly overestimated when the procedure was limited to solvent extraction. Conversely, the third procedure proved the efficiency of the chromatographic step since specificity was greatly improved in the three cases, the levels obtained with either kits being similar to those of the reference technique. The second group of results (above 270 nmol/l) yielded by the three kits were not always higher than those of HPLC when the procedure was limited to solvent extraction. When column chromatography was included in the procedure, the results were comparable to those of HPLC in three cases and lower in the three others. Since, the latter samples were collected after cortisol administration, and overestimated cortisol values obtained by HPLC might be due to the interference of some cortisol metabolites.     

Production of anti-idiotype antibodies for deoxynivalenol and their evaluation with three immunoassay platforms

Immunoassays for deoxynivalenol (DON) that involve binding to DON-specific antibodies have been widely developed. In such assays, the responses of samples are generally compared with calibration curves generated by using DON in competition with labeled reagents such as enzymatic or fluorescent conjugates of the toxin. However, materials that mimic the toxin can also be used, provided that they compete effectively with the labeled reagents for the DON-specific antibodies. Examples include certain types of anti-idiotype antibodies, obtained by the immunization of animals with toxin-specific antibodies. In the present work, anti-idiotype antibodies were developed which mimicked DON in the ability to bind to a DON-specific monoclonal antibody (Mab). Fab fragments of the Mab (Ab1) were used to immunize rabbits. Sera were screened by competitive direct enzyme linked immunosorbent assay (CD-ELISA) for the presence of anti-idiotype antibodies (Ab2). In order to determine the most effective screening format and also the potential efficacy in various forms of biosensors, the sera were further evaluated in biolayer interferometry (BLI) and fluorescence polarization immunoassay (FPIA) formats. All three formats were used to demonstrate the presence of anti-idiotypes capable of binding to the paratope of the DON antibody (subtypes Ab2β or Ab2γ). Such materials have the potential to replace DON as calibrants in immunoassays for this toxin.     

Comprehensive assessment of array-based platforms and calling algorithms for detection of copy number variants

We have systematically compared copy number variant (CNV) detection on eleven microarrays to evaluate data quality and CNV calling, reproducibility, concordance across array platforms and laboratory sites, breakpoint accuracy and analysis tool variability. Different analytic tools applied to the same raw data typically yield CNV calls with <50% concordance. Moreover, reproducibility in replicate experiments is <70% for most platforms. Nevertheless, these findings should not preclude detection of large CNVs for clinical diagnostic purposes because large CNVs with poor reproducibility are found primarily in complex genomic regions and would typically be removed by standard clinical data curation. The striking differences between CNV calls from different platforms and analytic tools highlight the importance of careful assessment of experimental design in discovery and association studies and of strict data curation and filtering in diagnostics. The CNV resource presented here allows independent data evaluation and provides a means to benchmark new algorithms.     

Rhinoviruses infect human epithelial cells via ceramide-enriched membrane platforms

The cell membrane contains very small distinct membrane domains enriched of sphingomyelin and cholesterol that are named rafts. We have shown that the formation of ceramide via activation of the acid sphingomyelinase transforms rafts into ceramide-enriched membrane platforms. These platforms are required for infection of mammalian cells with Pseudomonas aeruginosa, Staphylococcus aureus, or Neisseriae gonorrhoeae. In the present study we determined whether the acid sphingomyelinase, ceramide, and ceramide-enriched membrane platforms are also involved in the infection of human cells with pathogenic rhinoviruses. We demonstrate that infection of human epithelial cells with several rhinovirus strains triggers a rapid activation of the acid sphingomyelinase correlating with microtubules- and microfilament-mediated translocation of the enzyme from an intracellular compartment onto the extracellular leaflet of the cell membrane. The activity of the acid sphingomyelinase results in the formation of ceramide in the cell membrane and, finally, large ceramide-enriched membrane platforms. Rhinoviruses colocalize with ceramide-enriched membrane platforms during the infection. The significance of ceramide-enriched membrane platforms for rhinoviral uptake is demonstrated by the finding that genetic deficiency or pharmacological inhibition of the acid sphingomyelinase prevented infection of human epithelial cells by rhinoviruses. The data identify the acid sphingomyelinase and ceramide as key molecules for the infection of human cells with rhinoviruses.     

Predicting human chronically paralyzed muscle force: a comparison of three mathematical models.

Chronic spinal cord injury (SCI) induces detrimental musculoskeletal adaptations that adversely affect health status, ranging from muscle paralysis and skin ulcerations to osteoporosis. SCI rehabilitative efforts may increasingly focus on preserving the integrity of paralyzed extremities to maximize health quality using electrical stimulation for isometric training and/or functional activities. Subject-specific mathematical muscle models could prove valuable for predicting the forces necessary to achieve therapeutic loading conditions in individuals with paralyzed limbs. Although numerous muscle models are available, three modeling approaches were chosen that can accommodate a variety of stimulation input patterns. To our knowledge, no direct comparisons between models using paralyzed muscle have been reported. The three models include 1) a simple second-order linear model with three parameters and 2) two six-parameter nonlinear models (a second-order nonlinear model and a Hill-derived nonlinear model). Soleus muscle forces from four individuals with complete, chronic SCI were used to optimize each model's parameters (using an increasing and decreasing frequency ramp) and to assess the models' predictive accuracies for constant and variable (doublet) stimulation trains at 5, 10, and 20 Hz in each individual. Despite the large differences in modeling approaches, the mean predicted force errors differed only moderately (8-15% error; P=0.0042), suggesting physiological force can be adequately represented by multiple mathematical constructs. The two nonlinear models predicted specific force characteristics better than the linear model in nearly all stimulation conditions, with minimal differences between the two nonlinear models. Either nonlinear mathematical model can provide reasonable force estimates; individual application needs may dictate the preferred modeling strategy.