Digital Polymerase Chain Reaction Analysis Research Articles

A Versatile Microchannel Array Device for Portable and Parallel Droplet Generation

The efficient generation of monodispersed droplets holds great promise for micro‐/nanoparticle synthesis and biochemical analysis. However, it remains a challenge to achieve high‐throughput generation of monodispersed droplets in a portable and/or parallel manner in nonexpert biomedical laboratories. Herein, a versatile microchannel array (μCA) device is reported that is portable, multifaceted, reliable, and mass‐manufacturable, supporting the high‐throughput generation of monodispersed droplets in different ways. This device consists of a silicon‐based μCA chip based on the step emulsification principle, as well as two matching plastic containers, both of which can be mass‐manufactured by traditional microfabrication methods at low material costs. With the μCA device, aqueous solution can be dispersed into emulsion droplets by various modes, such as mechanical pump‐based large‐scale, handheld syringe‐based portable, and gas pump‐based highly parallel droplet generation. Furthermore, it is demonstrated that our cost‐effective device can be applied for digital polymerase chain reaction analysis, supporting the need for more accessible microfluidic systems. Thus, the present device is expected to have a significant impact on both benchside and bedside applications.

Facile prepared microfluidic chip for multiplexed digital RT-qPCR test.

The chip-based digital polymerase chain reaction (PCR) is an indispensable technique for amplifying and quantifying nucleic acids, which has been widely employed in molecular diagnostics at both fundamental and clinical levels. However, the previous designs have yet to achieve widespread application due to limitations in complex chip fabrication, pretreatment procedures, special surface properties, and low throughput. This study presents a facile digital microfluidic chip driven by centrifugal force for digital PCR analysis. Interestingly, regardless of the hydrophilicity or hydrophobicity of the inner chip surface, an efficient digitization process can be achieved. PCR reagents introduced into the inlet can be allocated to 9600 microchambers and subsequently isolated by the immiscible phase (silicone oil). The centrifugal priming approach offers a facile means to achieve high-throughput analysis. The design was further employed for the quantification of nucleic acids using digital PCR. The calculated result exhibited a strong correlation with the measured value at the concentrations from 1 copy/μL to 1000 copies/μL (R2 =0.99). Additionally, the chip also allowed digital multiplexed analysis, thereby indicating its potential for multi-target detection applications.

Atypical Droplet Digital Polymerase Chain Reaction Patterns That Indicate Uncommon but Clinically Actionable EGFR Mutations in Lung Cancer.

Droplet digital polymerase chain reaction (ddPCR) is a sensitive method to detect common pathogenic EGFR mutations in non-small cell lung cancer. Although targeted assays have not been specifically designed to detect them, uncommon EGFR mutations have been linked to response to targeted therapy. To describe atypical ddPCR patterns that correspond to uncommon but clinically actionable EGFR mutations. A cohort of 1134 consecutive non-small cell lung cancers that underwent targeted next-generation sequencing was reviewed. Uncommon EGFR mutations involving probe binding sites were evaluated by ddPCR. Two hundred fifty-five of 1134 cancers (22.5%) harbored pathogenic EGFR mutations. One hundred eighty-six of 255 (72.9%) had canonical EGFR exon 19 deletion or exon 21 p.L858R variants designed for detection by ddPCR. An additional 25 of 255 cases (9.8%) had uncommon EGFR mutations within the probe-binding site, including 1 case with concurrent uncommon mutations in both exon 19 and exon 21. These mutations included uncommon EGFR exon 19 deletions (n = 6), EGFR exon 19 substitutions p.L747P (n = 3) and p.L747A (n = 1), dinucleotide substitutions leading to EGFR p.L858R (n = 5), EGFR exon 21 substitutions p.K860I (n = 1) and p.L861Q (n = 9), and EGFR p.[L858R;K860I] (n = 1). Droplet digital polymerase chain reaction generated atypical but reproducible signal for each of these uncommon variants. Droplet digital polymerase chain reaction analysis of uncommon pathogenic EGFR variants can yield unique and reproducible results. Recognition of atypical patterns in EGFR ddPCR testing can prompt confirmatory molecular testing and aid appropriate targeted therapy selection for patients with non-small cell lung cancer.

Precise, wide field, and low-cost imaging and analysis of core-shell beads for digital polymerase chain reaction.

Digital droplet reactors have become a valuable tool for the analysis of single cells, organisms, or molecules by discretising reagents into picolitre or nanolitre volumes. However, DNA-based assays typically require processing of samples on the scale of tens of microlitres, with the detection of as few as one or as many as a hundred thousand fragments. Through the present work, we introduce a flow-focusing microfluidic device that produces 120 picolitre core-shell beads, which are assembled into a monolayer in a Petri dish for visualization and analysis. The bead assembly is subjected to polymerase chain reaction (PCR) amplification and fluorescence detection to digitally quantify the DNA concentration of the sample. We use a low-cost 21-megapixel digital camera and macro lens to capture wide-field fluorescence images with a 10-30 mm2 field-of-view at magnifications ranging from 5× to 2.5×. A customised Python script analysed the acquired images. Our study demonstrates the ability to perform digital PCR analysis of the entire bead assembly through end-point imaging and compare the results with those obtained through RT-qPCR.

Bead-Based Multiplexed Droplet Digital Polymerase Chain Reaction in a Single Tube Using Universal Sequences: An Ultrasensitive, Cross-Reaction-Free, and High-Throughput Strategy.

The multiplexed digital polymerase chain reaction (PCR) is widely used in molecular diagnosis owing to its high sensitivity and throughput for multiple target detection compared with the single-plexed digital PCR; however, current multiplexed digital PCR technologies lack efficient coding strategies that do not compromise the sensitivity and signal-to-noise (S/N) ratio. Hence, we propose a fluorescent-encoded bead-based multiplexed droplet digital PCR method for ultra-high coding capacity, along with the creative design of universal sequences (primer and fluorescent TaqMan probe) for ultra-sensitivity and high S/N ratios. First, pre-amplification is used to introduce universal primers and universal fluorescent TaqMan probes to reduce primer interference and background noise, as well as to enrich regions of interest in targeted analytes. Second, fluorescent-encoded beads (FEBs), coupled with the corresponding target sequence-specific capture probes through streptavidin-biotin conjugation, are used to partition amplicons via hybridization according to the Poisson distribution. Finally, FEBs mixed with digital PCR mixes are isolated into droplets generated via Sapphire chips (Naica Crystal Digital PCR system) to complete the digital PCR and result analysis. For proof of concept, we demonstrate that this method achieves high S/N ratios in a 5-plexed assay for influenza viruses and SARS-CoV-2 at concentrations below 10 copies and even close to a single molecule per reaction without cross-reaction, further verifying the possibility of clinical actual sample detection with 100% accuracy, which paves the way for the realization of digital PCR with ultrahigh coding capacity and ultra-sensitivity.

The respiratory microbiome associated with chronic obstructive pulmonary disease comorbidity in non-small cell lung cancer.

BackgroundResearch has shown that some microbiomes are linked to cancer. Hence, we hypothesize that alterations in the respiratory microbiome might be associated with lung cancer.MethodsThrough droplet digital polymerase chain reaction analysis, we investigated the abundance of Acidovorax in surgically resected primary tumors and corresponding nontumor lung tissues obtained from 50 Japanese patients with non‐small cell lung cancer.ResultsThe rate of positivity for Acidovorax in tumor and nontumor tissues was 44 and 26%, respectively. The abundance of Acidovorax in tumor tissues was significantly higher in patients with nonsquamous cell carcinoma complicated by chronic obstructive pulmonary disease (COPD) and those who relapsed after surgical resection (p < 0.05). In tumor tissues, the results of the univariate and multivariate analyses revealed that only COPD exerted a direct effect on the abundance of Acidovorax (p < 0.05). Furthermore, the presence of Acidovorax was high in lung cancer patients with COPD comorbidity (65%) and TP53 gene mutation; only one of the nontumor tissues was positive for Acidovorax. In patients with lung cancer complicated by COPD, Acidovorax tended to be present in both the tumor and nontumor areas.ConclusionsThis study identified novel microbiota involved in lung cancer with COPD comorbidity. The results suggested that Acidovorax may be a useful biomarker in the screening for lung cancer. Further studies are warranted to validate the clinical significance of the microbiome in a larger independent patient cohort.

The development of real-time digital PCR technology using an improved data classification method

For digital polymerase chain reaction (PCR), data classification is always a crucial task. The dynamic real-time amplification process information of each partition is always ignored in typical digital PCR analysis, which can easily lead to inaccurate outcomes. In this work, an integrated device that offers real-time chip-based digital PCR analysis was established. In addition, an enhanced process-based classification model (PAM) was built and trained. And then the device and the analytical model were employed in classification tasks for different concentrations of Epstein-Barr Virus (EBV) plasmid quantification assays. The results indicated that the real-time analysis device achieved a linearity of 0.97, the classification method was able to distinguish the false-positive curves, and the recognition error of positive wells was decreased by 64.4% compared with typical static analysis techniques when low concentrations of samples were tested. With these advantages, it is supposed that the real-time digital PCR analysis apparatus and the improved classification method can be employed to enhance the performance of digital PCR technology.

The Potential of Digital Polymerase Chain Reaction for Improving Diagnostic Yield of Nontuberculous Mycobacteria Pulmonary Disease.

IntroductionMany patients with nontuberculous mycobacteria pulmonary disease are asymptomatic. The disease diagnosis is confirmed in only a small proportion of patients with radiological findings suspicious for nontuberculous mycobacteria pulmonary disease. Thus, many patients remained undiagnosed. Here, we evaluated the diagnostic value of digital polymerase chain reaction (PCR) in nontuberculous mycobacteria pulmonary disease.MethodsWe prospectively evaluated 123 patients with radiological findings suspicious for nontuberculous mycobacteria pulmonary disease. Digital PCR was performed using bronchial lavage fluid, sputum, saliva, blood, and urine.ResultsThe culture of bronchial washing fluid was positive for nontuberculous mycobacteria in 53 patients and negative in 70. The positive detection rate of nontuberculous mycobacteria by digital PCR in patients with positive culture (n = 53) was as follows: bronchial lavage fluid 100%, sputum 62.9%, saliva 41.5%, blood 7.5%, and urine 3.8%. All patients with two or more positive partitions for nontuberculous mycobacteria in the digital PCR of bronchial lavage fluid showed nontuberculous mycobacteria growth in the bronchial lavage fluid culture. The digital PCR analysis of the bronchial lavage fluid showed a high sensitivity (100%), specificity (85.7%), positive predictive value (84.1%), negative predictive value (100%), and a high concordance rate (91.9%) with the bronchial lavage fluid culture results. In addition, the culture of bronchial lavage fluid was positive for nontuberculous mycobacteria in patients with two or more positive partitions in the digital PCR of sputum and saliva with a combined positive predictive value of 81.1%.ConclusionDigital PCR analysis of nontuberculous mycobacteria in bronchial lavage fluid shows a high concordance rate with the bronchial lavage fluid culture results and a high positive predictive value using both sputum and saliva, suggesting the potential usefulness of dPCR for diagnosis of nontuberculous mycobacteria pulmonary disease in clinical practice.

Case Report: Identification of Maternal Low-Level Mosaicism in the Dystrophin Gene by Droplet Digital Polymerase Chain Reaction

Germline mosaicism should be suspected when the same de novo mutations are identified in a second pregnancy with asymptomatic parents. Our study aims to find a feasible approach to reveal the existence of germline mosaicism. Multiplex Ligation-dependent Probe Amplification was performed on a Duchenne muscular dystrophy affected pedigree to detect deletion mutations. Then gap-polymerase chain reaction was performed to amplify the breakpoints junction sequence. Droplet digital polymerase chain reaction was utilized to identify the mutation frequencies in healthy parents. The same deletion in the exon 51 of the dystrophin gene, which was 50,035 bp in size, was detected in the proband and the fetus but not in their parents. Droplet digital polymerase chain reaction analysis of peripheral blood samples revealed mutant alleles of 3.53% in maternal blood cells. We here report a case of maternal low-level mosaicism confirmed by droplet digital polymerase chain reaction in peripheral blood samples, which reveals the existence of germline mosaicism. Gap-polymerase chain reaction combined with droplet digital polymerase chain reaction provide insights into the detection of germline mosaicism.

Mixed ductal-lobular carcinoma: an analysis of CDH1 DNA copy number variation and mutation.

Mixed ductal-lobular carcinoma (MDL) of the breast is poorly understood. Dysfunction of E-cadherin, a cell adhesion protein encoded by the CDH1 gene located on 16q22.1, causes loss of cell adhesion and cellular polarity in lobular carcinoma (LC). This study focuses the aberrations of CDH1 in LC, ductal carcinoma (DC), and MDL to investigate the pathogenesis of MDL. The CDH1 DNA value (ratio of CDH1 copy number to the reference gene, RNase P) was calculated by digital polymerase chain reaction analysis of a total of 113 breast carcinoma cases (51 LCs, 54 DCs, and 8 MDLs). CDH1 gene mutation assay was performed for 20/51 LCs, 8/54 DCs, and 8 MDLs cases. The CDH1 DNA values were lower in LCs (average: 0.664) than in DCs (average: 1.296) (p < 0.000). In MDL, The CDH1 DNA values were significantly lower in LC areas (average: 0.58), compared to that of DC areas (average: 1.08) (p = 0.004), and there is no significant difference between the intermingled areas (average: 1.05) and DC areas (p = 0.775). Moreover, CDH1 mutations occurred more frequently in MDLs than in pure LCs and DCs. In one MDL case, the identical CDH1 mutation was found in LC and DC areas. Our study presented that MDL had more frequent CDH1 mutations. There were two possible processes for cancer cells in LC areas: one process was via DC areas with a common ancestor, and another was an independent process from DC areas.

Transcriptome analysis of a ring chromosome 20 patient cohort.

Ring chromosomes occur when the ends of normally rod-shaped chromosomes fuse. In ring chromosome 20 (ring 20), intellectual disability and epilepsy are usually present, even if there is no deleted coding material; the mechanism by which individuals with complete ring chromosomes develop seizures and other phenotypic abnormalities is not understood. We investigated altered gene transcription as a contributing factor by performing RNA-sequencing (RNA-seq) analysis on blood from seven patients with ring 20, and 11 first-degree relatives (all parents). Geographic analysis did not identify altered expression in peritelomeric or other specific chromosome 20 regions. RNA-seq analysis revealed 97 genes potentially differentially expressed in ring 20 patients. These included one epilepsy gene, NPRL3, but this finding was not confirmed on reverse transcription Droplet Digital polymerase chain reaction analysis. Molecular studies of structural chromosomal anomalies such as ring chromosome are challenging and often difficult to interpret because many patients are mosaic, and there may be genome-wide chromosomal instability affecting gene expression. Our findings nevertheless suggest that peritelomeric altered transcription is not the likely pathogenic mechanism in ring 20. Underlying genetic mechanisms are likely complex and may involve differential expression of many genes, the majority of which may not be located on chromosome 20.

KRAS Mutations in Papillary Fibroelastomas: A Study of 50 Cases With Etiologic and Diagnostic Implications.

Papillary fibroelastoma (PFE) is an increasingly recognized cardiac tumor. Despite its prevalence, controversy exists as to whether it represents a reactive or neoplastic process due to histopathologic similarities with Lambl excrescences (LEs), an accepted reactive phenomenon. Recently, KRAS mutations were reported in a small collection of PFEs, but the incidence of mutations and conditions in which they arise in are unknown. Furthermore, the relationship between PFE and LE has yet to be investigated. Institutional archives were queried for cases of PFE (2001-2017). Paraffin-embedded tissue was microdissected for tumor isolation. Prospectively identified LEs (2018) were collected and wholly isolated. Extracted DNA underwent droplet digital polymerase chain reaction analysis of the most common KRAS mutations (codons 12/13 and 61). Relevant clinical information was abstracted from the medical record. Fifty-two PFEs were tested from 50 patients (32 women). The median patient age was 67 years. Seventeen (33%) PFEs harbored pathogenic variants in tested KRAS codons (12 in codons 12/13; 5 in codon 61). Mutations were mutually exclusive. No clinical or pathologic correlates differed significantly from cases without detectable pathogenic variants. No pathogenic mutation were detected in LEs (n=20; P=0.002). Herein, we report on the largest series of PFE tested for KRAS mutations and present the largest cohort of KRAS-mutant PFEs to date, providing evidence in support of the notion that at least a subset of PFEs represents neoplasia. Moreover, the lack of KRAS mutations in LEs provides evidence as to the separate etiology of this accepted reactive lesion.

KRAS G12D or G12V Mutation in Human Brain Arteriovenous Malformations

Brain arteriovenous malformations (BAVMs) are vascular malformations composed of tangles of abnormally developed vasculature without capillaries. Abnormal shunting of arteries and veins is formed, resulting in high-pressure vascular channels, which potentially lead to rupture. BAVMs are generally considered a congenital disorder. But clinical evidence regarding involution, regrowth, and de novo formation argue against the static condition of this disease. Recently, the presence of the somatic activating KRAS mutations in more than half of BAVM cases was reported, suggesting the role of KRAS function in the pathogenesis. KRAS mutation in codon35 (G→A, G12D; G→T, G12V) was examined by a digital polymerase chain reaction analysis using genome purified from paraffin-embedded slides of human BAVMs. We also examined protein expression of KRAS G12D in lesions to corroborate results from digital polymerase chain reaction analysis. We detected codon35 G→A mutation in 15 (39.5%) among 38 samples and codon35 G→T mutation in 10 (27.0%) among 37 samples we could assess mutations. There were no samples positive for both codon35 G→A and G→T mutation. The ratio of codon35 G→A mutation ranged from 0.60% to 12.28% and that of G→T was from 1.20% to 8.99%. We next examined protein expression of KRAS G12D in BAVM lesions in immunohistochemistry. A KRAS G12D mutant was detected mainly in endothelial cells of dilated vessels in lesions. KRAS mutations in codon35 were detected in about two thirds of specimens examined. KRAS function may actively contribute to the pathobiology of BAVM and can become a therapeutic target.

Distinct developmental trajectories of endometriotic epithelium and stroma: implications for the origins of endometriosis.

Endometriosis is a common gynecological disease characterized by the ectopic growth of endometrium-like tissue. Despite the widespread prevalence of endometriosis, its pathogenesis remains poorly understood. A recent study by Noë et al provides evidence that the epithelium and stroma within the same endometriotic lesions follow distinct and independent developmental trajectories. They used droplet digital polymerase chain reaction analysis of laser-captured epithelium-enriched and stroma-enriched endometriosis tissue, and found that all 19 somatic passenger mutations analyzed were enriched exclusively in the epithelial compartment. These findings are consistent with the clonal expansion of epithelial cells, whereas stromal cells may be continuously regenerated or recruited over the course of disease. Further findings of differing allelic frequencies among passenger mutations within the epithelium of the same endometriotic lesions are suggestive of subclonality or the existence of multiple clones in some cases. Overall, the authors' observations of clonally dominant somatic passenger mutations in the epithelium and not the stroma of endometriosis add to the recent description of cancer-associated mutations in such lesions, and provide clues to the pathogenesis of endometriosis. Further studies to determine where and when these mutations occur and whether they can be used to develop the first biologically informed classification system for endometriosis are warranted. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Digital Polymerase Chain Reaction Quantification of SERPINA1 Predicts Prognosis in High-Grade Glioma

SERPINA1 plays an anti-inflammatory role in protecting tissues from proteolytic mechanisms. SERPINA1 is positive in gliomas by immunohistochemical analysis; however, the role of SERPINA1, including the relationship with prognosis, has been uncertain. In recent years, digital polymerase chain reaction (PCR) has provided ultra-sensitive assessment of messenger RNA expression from formalin-fixed paraffin-embedded (FFPE) tissues. In this study, we quantitatively determined the expression of SERPINA1 in high-grade gliomas (HGGs) using digital PCR, and we analyzed its relationship with prognosis. Twenty-nine FFPE surgical samples from patients with HGGs (7 of World Health Organization [WHO] grade III and 22 of WHO grade IV), and human glioblastoma cell lines, U87 and U118, were used for analysis. A qualitative assessment using immunostaining and quantitative assessment using digital PCR were performed to assess the expression of SERPINA1. The expression of SERPINA1 was demonstrated in glioma tissues and glioblastoma multiforme cell lines by immunostaining. Digital PCR analysis showed that SERPINA1 was expressed in 14.3% and 63.6% of the tissues from patients with grade III and grade IV HGG, respectively (P= 0.035). The median overall survival of 38.8 months in the low SERPINA1 expression group was longer than that of 15.3 months in the high expression group (P= 0.030). The frequency and the amount of SERPINA1 expression were higher in grade IV than in grade III HGGs. The high expression of SERPINA1 indicates a poor prognosis of HGGs.

Genetic stability of Rift Valley fever virus MP-12 vaccine during serial passages in culture cells

Rift Valley fever is a mosquito-borne zoonotic disease endemic to Africa, which affects both ruminants and humans. Rift Valley fever causes serious damage to the livestock industry and is also a threat to public health. The Rift Valley fever virus has a segmented negative-stranded RNA genome consisting of Large (L)-segment, Medium (M)-segment, and Small (S)-segment. The live-attenuated MP-12 vaccine is immunogenic in livestock and humans, and is conditionally licensed for veterinary use in the US. The MP-12 strain encodes 23 mutations (nine amino acid substitutions) and is attenuated through a combination of mutations in the L-segment, M-segment, and S-segment. Among them, the M-U795C, M-A3564G, and L-G3104A mutations contribute to viral attenuation through the L-segment and M-segment. The M-U795C, M-A3564G, L-U533C, and L-G3750A mutations are also independently responsible for temperature-sensitive phenotype. We hypothesized that a serial passage of the MP-12 vaccine in culture cells causes reversions of the MP-12 genome. The MP-12 vaccine and recombinant rMP12-ΔNSs16/198 were serially passaged 25 times. Droplet digital polymerase chain reaction analysis revealed that the reversion occurred at L-G3750A during passages of MP-12 in Vero or MRC-5 cells. The reversion also occurred at M-A3564G and L-U533C of rMP12-ΔNSs16/198 in Vero cells. Reversion mutations were not found in MP-12 or the variant, rMP12-TOSNSs, in the brains of mice with encephalitis. This study characterized genetic stability of the MP-12 vaccine and the potential risk of reversion mutation at the L-G3750A temperature-sensitive mutation after excessive viral passages in culture cells.

ENESTgoal Treatment-Free Remission Study: Updated Preliminary Results and Digital Polymerase Chain Reaction Analysis in Patients with Chronic Myeloid Leukemia in Chronic Phase Who Switched from Imatinib to Nilotinib

ENESTgoal Treatment-Free Remission Study: Updated Preliminary Results and Digital Polymerase Chain Reaction Analysis in Patients with Chronic Myeloid Leukemia in Chronic Phase Who Switched from Imatinib to Nilotinib

MYEOV gene overexpression in primary plasma cell leukemia with t(11;14)(q13;q32).

Primary plasma cell leukemia (pPCL) is an uncommon form of plasma cell dyscrasia, and the most aggressive of the human monoclonal gammopathies. The t(11;14)(q13;q32) rearrangement is the most common alteration in pPCL, promoting cyclin D1 (CCND1) gene overexpression caused by its juxtaposition with the immunoglobulin heavy locus chromosome region. The myeloma overexpressed (MYEOV) gene maps very close to the CCND1 gene on chromosome 11, but its overexpression is rarely observed in multiple myeloma. The present study describes a case of pPCL with t(11;14) characterized by a breakpoint on der(11), unlike the one usually observed. Droplet digital polymerase chain reaction analysis revealed overexpression of CCND1 and MYEOV. To the best of our knowledge, MYEOV gene overexpression has never been previously described in pPCL.

Developments in precision medicine: Studies highlight new applications for cancer chemoprevention, treatment.

Developments in precision medicine: Studies highlight new applications for cancer chemoprevention, treatment.

The nitrogen cycle in cryoconites: naturally occurring nitrification-denitrification granules on a glacier.

Cryoconites are microbial aggregates commonly found on glacier surfaces where they tend to take spherical, granular forms. While it has been postulated that the microbes in cryoconite granules play an important role in glacier ecosystems, information on their community structure is still limited, and their functions remain unclear. Here, we present evidence for the occurrence of nitrogen cycling in cryoconite granules on a glacier in Central Asia. We detected marker genes for nitrogen fixation, nitrification and denitrification in cryoconite granules by digital polymerase chain reaction (PCR), while digital reverse transcription PCR analysis revealed that only marker genes for nitrification and denitrification were abundantly transcribed. Analysis of isotope ratios also indicated the occurrence of nitrification; nitrate in the meltwater on the glacier surface was of biological origin, while nitrate in the snow was of atmospheric origin. The predominant nitrifiers on this glacier belonged to the order Nitrosomonadales, as suggested by amoA sequences and 16S ribosomal RNA pyrosequencing analysis. Our results suggest that the intense carbon and nitrogen cycles by nitrifiers, denitrifiers and cyanobacteria support abundant and active microbes on the Asian glacier.