Rapid evaluation and quality control of next generation sequencing data with FaQCs

Background

Next generation sequencing (NGS) technologies that parallelize the sequencing process and produce thousands to millions, or even hundreds of millions of sequences in a single sequencing run, have revolutionized genomic and genetic research. Because of the vagaries of any platform’s sequencing chemistry, the experimental processing, machine failure, and so on, the quality of sequencing reads is never perfect, and often declines as the read is extended. These errors invariably affect downstream analysis/application and should therefore be identified early on to mitigate any unforeseen effects.

Results

Here we present a novel FastQ Quality Control Software (FaQCs) that can rapidly process large volumes of data, and which improves upon previous solutions to monitor the quality and remove poor quality data from sequencing runs. Both the speed of processing and the memory footprint of storing all required information have been optimized via algorithmic and parallel processing solutions. The trimmed output compared side-by-side with the original data is part of the automated PDF output. We show how this tool can help data analysis by providing a few examples, including an increased percentage of reads recruited to references, improved single nucleotide polymorphism identification as well as de novo sequence assembly metrics.

Conclusion

FaQCs combines several features of currently available applications into a single, user-friendly process, and includes additional unique capabilities such as filtering the PhiX control sequences, conversion of FASTQ formats, and multi-threading. The original data and trimmed summaries are reported within a variety of graphics and reports, providing a simple way to do data quality control and assurance.

Electronic supplementary material

The online version of this article (doi:10.1186/s12859-014-0366-2) contains supplementary material, which is available to authorized users.     

Paired analysis of tumor mutation burden calculated by targeted deep sequencing panel and whole exome sequencing in non-small cell lung cancer

Owing to rapid advancements in NGS (next generation sequen-cing), genomic alteration is now considered an essential pre-dictive biomarkers that impact the treatment decision in many cases of cancer. Among the various predictive biomarkers, tumor mutation burden (TMB) was identified by NGS and was con-sidered to be useful in predicting a clinical response in cancer cases treated by immunotherapy. In this study, we directly com-pared the lab-developed-test (LDT) results by target sequencing panel, K-MASTER panel v3.0 and whole-exome sequencing (WES) to evaluate the concordance of TMB. As an initial step, the reference materials (n = 3) with known TMB status were used as an exploratory test. To validate and evaluate TMB, we used one hundred samples that were acquired from surgically resected tissues of non-small cell lung cancer (NSCLC) patients. The TMB of each sample was tested by using both LDT and WES methods, which extracted the DNA from samples at the same time. In addition, we evaluated the impact of capture re-gion, which might lead to different values of TMB; the evalu-ation of capture region was based on the size of NGS and target sequencing panels. In this pilot study, TMB was evalu-ated by LDT and WES by using duplicated reference samples; the results of TMB showed high concordance rate (R² = 0.887). This was also reflected in clinical samples (n = 100), which showed R² of 0.71. The difference between the coding sequence ratio (3.49%) and the ratio of mutations (4.8%) indicated that the LDT panel identified a relatively higher number of mutations. It was feasible to calculate TMB with LDT panel, which can be useful in clinical practice. Furthermore, a customized approach must be developed for calculating TMB, which differs according to cancer types and specific clinical settings.     

一种基于基因拓扑重要性的通路识别方法

癌症相关通路的识别是认识癌症发生发展过程机制的生物学基础。已有的通路识别方法很少考虑基因在通路中的拓扑重要性。重叠基因降权(PADOG)方法在基因集分析(GSA)方法的基础上融入了基因特异性的影响,提高了癌症相关通路的识别性能。为进一步提高癌症相关通路的识别性能,首先统计了KEGG通路数据集中基因出度的分布情况,根据基因出度的大小定义了基因的重要性。最后将基因的特异性和重要性融合在一起,提出了一种基于基因重要性和特异性的通路分析方法 PAGIS。在结肠癌、肺癌和胰腺癌3个数据集上的实验结果表明,PAGIS方法比PADOG能够提高很多癌症相关的排名,从而提高癌症相关通路的识别效果。     

Exploring the mechanism of ellagic acid against gastric cancer based on bioinformatics analysis and network pharmacology

Ellagic acid (EA) is a natural polyphenolic compound. Recent studies have shown that EA has potential anticancer properties against gastric cancer (GC). This study aims to reveal the potential targets and mechanisms of EA against GC. This study adopted methods of bioinformatics analysis and network pharmacology, including the weighted gene co-expression network analysis (WGCNA), construction of protein–protein interaction (PPI) network, receiver operating characteristic (ROC) and Kaplan–Meier (KM) survival curve analysis, Gene Ontology (GO) function and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis, molecular docking and molecular dynamics simulations (MDS). A total of 540 EA targets were obtained. Through WGCNA, we obtained a total of 2914 GC clinical module genes, combined with the disease database for screening, a total of 606 GC-related targets and 79 intersection targets of EA and GC were obtained by constructing Venn diagram. PPI network was constructed to identify 14 core candidate targets; TP53, JUN, CASP3, HSP90AA1, VEGFA, HRAS, CDH1, MAPK3, CDKN1A, SRC, CYCS, BCL2L1 and CDK4 were identified as the key targets of EA regulation of GC by ROC and KM curve analysis. The enrichment analysis of GO and KEGG pathways of key targets was performed, and they were mainly enriched in p53 signalling pathway, PI3K-Akt signalling pathway. The results of molecular docking and MDS showed that EA could effectively bind to 13 key targets to form stable protein–ligand complexes. This study revealed the key targets and molecular mechanisms of EA against GC and provided a theoretical basis for further study of the pharmacological mechanism of EA against GC.     

A bird's-eye view on the modern genetics workflow and its potential applicability to the locust problem

Genetics is an immense science and the current developments in its methods and techniques as well as the fast emerging tools make it one of the most powerful biological sciences. Indeed, from taxonomy and ecology to physiology and molecular biology, every biological science makes use of genetics techniques and methods at one time or another. In fact, development in genetics is such that it is now possible to characterize and analyze the expression of the whole set of genes of virtually every living organism, even if it is a non-model one. Locusts are notorious for the damage they cause to the ecosystems and economies of the areas affected by their recurrent population outbreaks. To prevent and deal with these outbreaks, we now count on both biological as well as chemical agents that are proving to be successful in reducing the damage that otherwise locust population outbreaks might cause. However, a better, efficient and environmentally friendly solution is still a hoped-for target. In my opinion, the ideal future pesticide should be both environmentally friendly, risk free and species-specific. To reach the knowledge needed for the development of such species-specific anti-locust agent, deep and accurate knowledge of the locusts’ genetics and molecular biology is a must. Since genes and their expression levels lie at the bottom of every biological phenomenon, any species-specific solution to the locust problem requires a good knowledge of these organisms’ genes as well as the quantitative and spatio-temporal dynamics of their expression. To reach such knowledge, collaborative work is needed as well as a clear workflow that, given the fast development in the genetics tools, is not always clear to all research groups. For this reason, here I describe a genetics workflow that should allow taking advantage of the most recent genetics tools and techniques to answer question relating to locust biology. My hope is that the adoption of this and other work strategies by different research groups, especially when the work is a collaborative one, would provide precious information on the biology and the biological phenomena that these economically important organisms exhibit.     

Tissue transglutaminase induces the release of apoptosis inducing factor and results in apoptotic death of pancreatic cancer cells

Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal malignant disease with poor long-term survival rates. Major reason for poor disease outcome is the profound intrinsic resistance of PDAC cells to currently available treatment regimens. We recently found that a great majority of PDAC tumors and tumor cell lines express high basal level of tissue transglutaminase (TG2), a multifunctional protein implicated in apoptosis, cell attachment, cell survival, and cell motility functions. Based on these observations, we hypothesized that activation of endogenous TG2 can induce spontaneous apoptosis in PDAC cells. The results obtained suggested that activation of endogenous TG2 by calcium ionophore A23187 induced rapid and spontaneous apoptosis in PDAC cells. TG2-induced apoptosis was associated with release of apoptosis-inducing factor (AIF). The release of AIF from mitochondria led to its translocation to the nucleus and subsequent apoptosis of PDAC cells in caspase-independent manner. In conclusion, our results provide first evidence that TG2 can induce apoptosis in PDAC cells in an AIF-dependent and caspase-independent manner.     

Next generation sequencing as a useful tool in the diagnostics of mosaicism in Alport syndrome

Alport syndrome (ATS) is a progressive hereditary nephropathy characterized by hematuria and/or proteinuria with structural defects of the glomerular basement membrane. It can be associated with extrarenal manifestations (high-tone sensorineural hearing loss and ocular abnormalities). Somatic mutations in COL4A5 (X-linked), COL4A3 and COL4A4 genes (both autosomal recessive and autosomal dominant) cause Alport syndrome. Somatic mosaicism in Alport patients is very rare. The reason for this may be due to the difficulty of detection.     

The role of Algerian Ephedra alata ethanolic extract in inhibiting the growth of breast cancer cells by inducing apoptosis in a p53- dependent pathway

BackgroundEphedra alata, a member of the Ephedraceae family, was used to treat different diseases and it might be shown a strong efficacy to inhibit cancer cell lines.MethodsDue to the limited research available about this plant, the objective of this research was to evaluate the antioxidant, cytotoxic and apoptotic effects of Ephedra alata ethanolic extract (EAEE), against different human cancer cell lines.ResultsEAEE inhibited the growth of the liver (HepG2), breast (MCF-7), and colon cancer cells (Caco-2). MCF-7 cells with an IC₅₀ of 153 µg/ml, were the most sensitive to the extract. Furthermore, exploration using flow cytometry using Annexin V-FITC/PI assay demonstrated that EAEE caused death for all human cancer cells mainly through apoptosis. Very interestingly, qRT-PCR analysis using the ΔΔCt method revealed that four genes, Bax, p21, RB1, and TP53 were up-regulated in MCF-7 cells treated either with EAEE or S-FU drug. These findings let us believe that the mechanism by which EAEE kills breast cancer cells seems to be apoptosis via a P53-dependent manner, which involved intrinsic pathways through the induction of Bax, p21, and RB1.ConclusionsEAEE exhibits good biological properties in contradiction of HepG-2, MCF-7, and Caco-2 cell lines. This study appoints for the first time that EAEE increases the expression in MCF-7 cells of p53 and three more genetic traits that control cellular proliferation and apoptosis. Therefore, this plant could serve as a potential source to find new pro-apoptotic drugs for cancer treatment.     

miR-21的高表达与胰腺癌预后关系的meta分析

为评价miR-21的高表达与胰腺癌预后的相关性,通过全面检索Pub Med,EMBASE,Google scholar,维普,CNKI等数据库,收集已公开发表的关于miR-21的高表达与胰腺癌预后相关性的文献,按meta分析的要求对原始文献的质量进行评估,采用STATA V12.0软件对各研究的效应量进行统计分析。结果发现共纳入4篇文献(共300个病例),合并总生存率(OS)HR为1.26(95%CI:1.08~1.47,P0.05)。由此可以推断胰腺癌预后的一个危险因素为miR-21的高表达。     

Oleanolic acid increases the anticancer potency of doxorubicin in pancreatic cancer cells

Combination therapy is a novel cancer therapy approach that combines two or more chemotherapy drugs. This treatment modality enhances the efficacy of chemotherapy by targeting key pathways in an additive or synergistic manner. Therefore, we investigated the efficacy of combination therapy by widely used chemotherapy drug doxorubicin (DOX) and oleanolic acid (OA) to induction of apoptosis for pancreatic cancer (PC) therapy. The effects of DOX, OA, and their combination (DOX-OA) were investigated on proliferation and viability of PC cell line (PANC-1) by MTT assay. Moreover, migration and invasion of the cancer cells were evaluated by trans-well migration assay and wound healing assay. Flow cytometry and DAPI (4′,6-diamidino-2-phenylindole) staining were employed to investigate apoptosis quantification and qualification of the treated cancer cells. Finally, mRNA expression of apoptosis-related genes was assessed by quantitative real-time polymerase chain reaction. Our results demonstrated that the proliferation and metastasis potential of PC cells significantly decreased after treatment by DOX, OA, and DOX-OA. Moreover, we observed an increase in apoptosis percentage in the treated cancer cells. The apoptosis-related gene expression was modified to increase the apoptosis rate in all of the treatment groups. However, the anticancer potency of DOX-OA combination was significantly more than that of DOX and OA treatments alone. Our study suggested that DOX-OA combination exerts more profound anticancer effects against PC cell lines than DOX or OA monotherapy. This approach may increase the efficiency of chemotherapy and reduce unintended side effects by lowering the prescribed dose of DOX.     

MTA1 promotes the invasion and migration of pancreatic cancer cells potentially through the HIF-α/VEGF pathway

Abstract

The metastasis-associated gene 1 (MTA1) has previously been recognized as an oncogene, and abnormal MTA1 expression has been related to progression of numerous cancer types to the metastasis stage. However, the function of MTA1 in the regulation of pancreatic cancer progression and metastasis remains unclear. Western blot analysis was adopted to determine the expression of MTA1 in pancreatic cancer tissues and corresponding near normal tissues. Steady clone with MTA1-overexpression and MTA1-inhibitionweregenerated via lentivirus technology in BxPc-3 cells. Transwell assay was carried out for detecting the invasion of pancreatic cancer cells. The migration activity was assessed using the wound scratch assay. The effect of MTA1 in pancreatic cancer was evaluated in the mice xenografts. Western blot analysis was employed to determine the expression of hypoxia inducible factor-α (HIF-α) and vascular endothelial growth factor (VEGF) in vitro and in vivo. We observed that MTA1 overexpression enhanced migration and invasion ability of pancreatic cancer cells in vitro and increased HIF-α and VEGF protein levels in vitro and in vivo. MTA1 inhibition had the opposite effects. MTA1 protein level was positively related to HIF-α and VEGF protein levels. These results indicated that MTA1 potentially promoted pancreatic cancer metastasis via HIF-α/VEGF pathway. This research supplies a new molecular mechanism for MTA1 in the pancreatic cancer progression and metastasis. MTA1 may be an effective therapy target in pancreatic cancer.     

Nano-chemotherapy using cationic liposome that strategically targets the cell membrane potential of pancreatic cancer cells with negative charge

Negatively charged phosphatidylserine (PS) and sialic acid-containing glycosphingolipids (GM1) were observed to be over represented on the cell membranes of pancreatic cancer cells (BxPC-3) as opposed to normal pancreatic cells. Cationic liposomes (CL) were also found to selectively accumulate into the negatively charged cell membranes of BxPC-3 cells and inhibited their growth but have no effect on the viability of normal pancreatic cells. CL induced apoptosis in BxPC-3 cells via activation of caspase-3, -8, and -9 and mitochondrial events and inhibited tumor enlargement in xenograft mouse models of pancreatic cancer.     

In vitro and in vivo induction of apoptosis by capsaicin in pancreatic cancer cells is mediated through ROS generation and mitochondrial death pathway

Pancreatic cancer is one of the most common invasive malignancies and the fourth leading cause of cancer related mortality in U.S., thus developing new strategies to control pancreatic cancer is an important mission. We investigated the mechanism of capsaicin, the major pungent ingredient of red-chili pepper, in inducing apoptosis in pancreatic cancer cells. Treatment of AsPC-1 and BxPC-3 cells with capsaicin resulted in a dose-dependent inhibition of cell-viability and induction of apoptosis which was associated with the generation of ROS and persistent disruption of mitochondrial membrane potential. These effects were significantly blocked when the cells were pretreated with a general antioxidant N-acetyl cysteine (NAC). Exposure of AsPC-1 and BxPC-3 cells to capsaicin was also associated with increased expression of Bax, down-regulation of bcl-2, survivin and significant release of cytochrome c and AIF in the cytosol. On the contrary, above-mentioned effects were not observed in the normal acinar cells in response to capsaicin-treatment. Capsaicin-treatment resulted in the activation of JNK and JNK inhibitor SP600125 afforded protection against capsaicin-induced apoptosis. Furthermore, capsaicin when given orally markedly suppressed the growth of AsPC-1 pancreatic tumor xenografts in athymic nude mice, without side effects. Tumors from capsaicin treated mice demonstrated increased apoptosis, which was related to the activation of JNK and increased cytosolic protein expression of Bax, cytochrome c, AIF and cleaved caspase-3, as compared with controls. Taken together, these results show that capsaicin is an effective inhibitor of in vitro and in vivo growth of pancreatic cancer cells. These findings provide the rationale for further clinical investigation of capsaicin against pancreatic cancer. Ruifen Zhang and Ian Humphreys contributed equally to this work.     

Ultraviolet enhances the sensitivity of pancreatic cancer cells to gemcitabine by activation of 5' AMP-activated protein kinase

Although gemcitabine is recognized as the standard drug for the treatment of advanced pancreatic cancer, the clinical outcome is not satisfactory. We recently reported that relatively high dose ultraviolet-C (UV-C; 200 J) inhibits cell growth by desensitization of epidermal growth factor receptor (EGFR) in human pancreatic cancer cells. In the present study, we investigated the combination effects of low dose UV-C (10 J) and gemcitabine on apoptosis and cell growth in these cells. UV-C enhanced gemcitabine-induced suppression of cell viability. In addition, the combination use clearly induced apoptosis, while neither UV-C nor gemcitabine alone did. Concurrently, combination use caused the decrease in the EGFR protein level and reduced EGF-induced activation of Akt pathway, subsequently resulting in accumulation of β-catenin. The order of the treatment with UV-C and gemcitabine did not affect their synergistic effects on apoptosis and cell growth. Interestingly, combination use synergistically induced phosphorylation of 5′ AMP-activated protein kinase (AMPK) alpha at Thr172 and acetyl-CoA carboxylase at Ser79 as a downstream molecular target of AMPK. AMPK activator, 5-aminoimidazole-4-carboxamide-1-β-riboside, induced apoptosis and suppressed cell growth in these cells, thus suggesting that combination effects of UV-C and gemcitabine is due to the activation of AMPK. Together, our findings could provide a new aspect of pancreatic cancer therapy.     

Involvement of MEK-ERK signaling pathway in the inhibition of cell growth by troglitazone in human pancreatic cancer cells

Tif6p (eIF6) is necessary for 60S biogenesis, rRNA maturation and must be released from 60S to permit 80S assembly and translation. We characterized Tif6p interactors. Tif6p is mostly on 66S-60S pre-ribosomes, partly free. Tif6p complex(es) contain nucleo-ribosomal factors and Asc1p. Surprisingly, Tif6p particle contains the low-abundance endonuclease Sen34p. We analyzed Sen34p role on rRNA/tRNA synthesis, in vivo. Sen34p depletion impairs tRNA splicing and causes unexpected 80S accumulation. Accordingly, Sen34p overexpression causes 80S decrease and increased polysomes which suggest increased translational efficiency. With delayed kinetics, Sen34p depletion impairs rRNA processing. We conclude that Sen34p is absolutely required for tRNA splicing and that it is a rate-limiting element for efficient translation. Finally, we confirm that Tif6p accompanies 27S pre-rRNA maturation to 25S rRNA and we suggest that Sen34p endonuclease in Tif6p complex may affect also rRNA maturation.     

Identification and comparative analysis of the Eriocheir sinensis microRNA transcriptome response to Spiroplasma eriocheiris infection using a deep sequencing approach

The Chinese mitten crab Eriocheir sinensis is one of the most important freshwater aquaculture crustacean species in China. MicroRNAs (miRNAs) are small non-coding RNAs that are important effectors in the intricate host-pathogen interaction network. To increase the repertoire of miRNAs characterized in crustaceans and to examine the relationship between host miRNA expression and pathogen infection, we used the Illumina/Solexa deep sequencing technology to sequence two small RNA libraries prepared from haemocytes of E. sinensis under normal conditions and during infection with Spiroplasma eriocheiris. The high-throughput sequencing resulted in approximately 30,975,151 and 30,826,277 raw reads corresponding to 12,077,088 and 16,271,545 high-quality mappable reads for the normal and infected haemocyte samples, respectively. Bioinformatic analyses identified 735 unique miRNAs, including 36 that are conserved in crustaceans, 134 that are novel to crabs but are present in other arthropods (PN-type), and 565 that are completely new (PC-type). Two hundred twenty-eight unique miRNAs displayed significant differential expression between the normal and infected haemocyte samples (p < 0.0001). Of these, 133 (58%) were significantly up-regulated and 95 (42%) were significantly down-regulated upon challenge with S. eriocheiris. Real-time quantitative PCR (RT-qPCR) experiments were preformed for 10 miRNAs of the two samples, and agreement was found between the sequencing and RT-qPCR data. To our knowledge, this is the first report of comprehensive identification of E. sinensis miRNAs and of expression analysis of E. sinensis miRNAs after exposure to S. eriocheiris. Many miRNAs were differentially regulated when exposed to the pathogen, and these findings support the hypothesis that certain miRNAs might be essential in host-pathogen interactions. Our results suggest that elucidation of the molecular mechanisms responsible for miRNA regulation of the host’s innate immune system should help with the development of new control strategies to prevent or treat S. eriocheiris infections in crustaceans.