Amplicon-based Sequencing Approach Research Articles

Amplicon-Based Microbiome Profiling: From Second- to Third-Generation Sequencing for Higher Taxonomic Resolution.

The 16S rRNA amplicon-based sequencing approach represents the most common and cost-effective strategy with great potential for microbiome profiling. The use of second-generation sequencing (NGS) technologies has led to protocols based on the amplification of one or a few hypervariable regions, impacting the outcome of the analysis. Nowadays, comparative studies are necessary to assess different amplicon-based approaches, including the full-locus sequencing currently feasible thanks to third-generation sequencing (TGS) technologies. This study compared three different methods to achieve the deepest microbiome taxonomic characterization: (a) the single-region approach, (b) the multiplex approach, covering several regions of the target gene/region, both based on NGS short reads, and (c) the full-length approach, which analyzes the whole length of the target gene thanks to TGS long reads. Analyses carried out on benchmark microbiome samples, with a known taxonomic composition, highlighted a different classification performance, strongly associated with the type of hypervariable regions and the coverage of the target gene. Indeed, the full-length approach showed the greatest discriminating power, up to species level, also on complex real samples. This study supports the transition from NGS to TGS for the study of the microbiome, even if experimental and bioinformatic improvements are still necessary.

Development of an amplicon-based sequencing approach in response to the global emergence of mpox.

The 2022 multicountry mpox outbreak concurrent with the ongoing Coronavirus Disease 2019 (COVID-19) pandemic further highlighted the need for genomic surveillance and rapid pathogen whole-genome sequencing. While metagenomic sequencing approaches have been used to sequence many of the early mpox infections, these methods are resource intensive and require samples with high viral DNA concentrations. Given the atypical clinical presentation of cases associated with the outbreak and uncertainty regarding viral load across both the course of infection and anatomical body sites, there was an urgent need for a more sensitive and broadly applicable sequencing approach. Highly multiplexed amplicon-based sequencing (PrimalSeq) was initially developed for sequencing of Zika virus, and later adapted as the main sequencing approach for Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). Here, we used PrimalScheme to develop a primer scheme for human monkeypox virus that can be used with many sequencing and bioinformatics pipelines implemented in public health laboratories during the COVID-19 pandemic. We sequenced clinical specimens that tested presumptively positive for human monkeypox virus with amplicon-based and metagenomic sequencing approaches. We found notably higher genome coverage across the virus genome, with minimal amplicon drop-outs, in using the amplicon-based sequencing approach, particularly in higher PCR cycle threshold (Ct) (lower DNA titer) samples. Further testing demonstrated that Ct value correlated with the number of sequencing reads and influenced the percent genome coverage. To maximize genome coverage when resources are limited, we recommend selecting samples with a PCR Ct below 31 Ct and generating 1 million sequencing reads per sample. To support national and international public health genomic surveillance efforts, we sent out primer pool aliquots to 10 laboratories across the United States, United Kingdom, Brazil, and Portugal. These public health laboratories successfully implemented the human monkeypox virus primer scheme in various amplicon sequencing workflows and with different sample types across a range of Ct values. Thus, we show that amplicon-based sequencing can provide a rapidly deployable, cost-effective, and flexible approach to pathogen whole-genome sequencing in response to newly emerging pathogens. Importantly, through the implementation of our primer scheme into existing SARS-CoV-2 workflows and across a range of sample types and sequencing platforms, we further demonstrate the potential of this approach for rapid outbreak response.

Novel Amplicon-Based Sequencing Approach to West Nile Virus.

West Nile virus is a re-emerging arbovirus whose impact on public health is increasingly important as more and more epidemics and epizootics occur, particularly in America and Europe, with evidence of active circulation in Africa. Because birds constitute the main reservoirs, migratory movements allow the diffusion of various lineages in the world. It is therefore crucial to properly control the dispersion of these lineages, especially because some have a greater health impact on public health than others. This work describes the development and validation of a novel whole-genome amplicon-based sequencing approach to West Nile virus. This study was carried out on different strains from lineage 1 and 2 from Senegal and Italy. The presented protocol/approach showed good coverage using samples derived from several vertebrate hosts and may be valuable for West Nile genomic surveillance.

Bacterial, fungal, and interkingdom microbiome features of exclusively breastfeeding dyads are associated with infant age, antibiotic exposure, and birth mode.

The composition and function of early life gut bacterial communities (microbiomes) have been proposed to modulate health for the long term. In addition to bacteria, fungi (mycobiomes) also colonize the early life gut and have been implicated in health disorders such as asthma and obesity. Despite the potential importance of mycobiomes in health, there has been a lack of study regarding fungi and their interkingdom interactions with bacteria during infancy. The goal of this study was to obtain a more complete understanding of microbial communities thought to be relevant for the early life programming of health. Breastmilk and infant feces were obtained from a unique cohort of healthy, exclusively breastfeeding dyads recruited as part of the Mothers and Infants Linked for Healthy Growth (MILk) study with microbial taxa characterized using amplicon-based sequencing approaches. Bacterial and fungal communities in breastmilk were both distinct from those of infant feces, consistent with niche-specific microbial community development. Nevertheless, overlap was observed among sample types (breastmilk, 1-month feces, 6-month feces) with respect to the taxa that were the most prevalent and abundant. Self-reported antibacterial antibiotic exposure was associated with micro- as well as mycobiome variation, which depended upon the subject receiving antibiotics (mother or infant), timing of exposure (prenatal, peri- or postpartum), and sample type. In addition, birth mode was associated with bacterial and fungal community variation in infant feces, but not breastmilk. Correlations between bacterial and fungal taxa abundances were identified in all sample types. For infant feces, congruency between bacterial and fungal communities was higher for older infants, consistent with the idea of co-maturation of bacterial and fungal gut communities. Interkingdom connectedness also tended to be higher in older infants. Additionally, higher interkingdom connectedness was associated with Cesarean section birth and with antibiotic exposure for microbial communities of both breastmilk and infant feces. Overall, these results implicate infant age, birth mode, and antibiotic exposure in bacterial, fungal and interkingdom relationship variation in early-life-relevant microbiomes, expanding the current literature beyond bacteria.

Metagenomics and volatile profile of Turkish artisanal Tulum cheese microbiota

Turkish artisanal Tulum cheese is a traditional raw milk cheese and starter cultures are not used for ripening process. In this study, 16S rRNA amplicon-based sequencing approach was used for determining bacterial diversity in Tulum cheeses. The volatile organic compound (VOC) profile which contributes to sensory properties of the cheeses was determined via solid-phase microextraction combined with gas chromatography-mass spectrometry (GC-MS/SPME) and correlated with the microbiota. In Tulum cheese samples, Lactobacillaceae (30,71%) phylum widely varied but Streptococcaceae (59,86%) was dominant. It mainly belongs to the four dominant genera of lactic acid bacteria (LAB) and includes Lactobacillus, Lactococcus, Streptococcus and Pediococcus. The bacterial diversity of TC5 sample was much higher than that of other samples. Moreover, several species of non-starter LAB were frequently detected. A total of 36 volatile compounds were identified including hydrocarbons (3), acids (13), alcohols (5), ketones (3), aldehydes (3), esters (8) and miscellaneous compound (1). Pentadecanoic acid, octadecanoic acid, tetradecanoic acid, octanoic acid, hexanoic acid were determined to be dominant in all cheese samples and to be related to microbial flora. Also, the sensory profiles of the samples were examined. The most popular TC6 did not contain 2-butanone and hexanal from the volatile organic compound (VOC) values, while in the most unpopular TC3, 2-butanone (7.73%) and hexanal (5.95%) were found to be the highest. The outcome of this study will contribute to literature on bacterial diversity and aromatic profile of Tulum cheese which should be investigated in order to improve quality and safety.

In Vitro Reassortment between Endemic Bluetongue Viruses Features Global Shifts in Segment Frequencies and Preferred Segment Combinations.

Bluetongue virus (BTV) is an arthropod-borne pathogen that is associated with sometimes severe disease in both domestic and wild ruminants. Predominantly transmitted by Culicoides spp. biting midges, BTV is composed of a segmented, double-stranded RNA genome. Vector expansion and viral genetic changes, such as reassortment between BTV strains, have been implicated as potential drivers of ongoing BTV expansion into previously BTV-free regions. We used an in vitro system to investigate the extent and flexibility of reassortment that can occur between two BTV strains that are considered enzootic to the USA, BTV-2 and BTV-10. Whole genome sequencing (WGS) was coupled with plaque isolation and a novel, amplicon-based sequencing approach to quantitate the viral genetic diversity generated across multiple generations of in vitro propagation. We found that BTV-2 and BTV-10 were able to reassort across multiple segments, but that a preferred BTV-2 viral backbone emerged in later passages and that certain segments were more likely to be found in reassortant progeny. Our findings indicate that there may be preferred segment combinations that emerge during BTV reassortment. Moreover, our work demonstrates the usefulness of WGS and amplicon-based sequencing approaches to improve understanding of the dynamics of reassortment among segmented viruses such as BTV.

NOseq: amplicon sequencing evaluation method for RNA m6A sites after chemical deamination.

Methods for the detection of m6A by RNA-Seq technologies are increasingly sought after. We here present NOseq, a method to detect m6A residues in defined amplicons by virtue of their resistance to chemical deamination, effected by nitrous acid. Partial deamination in NOseq affects all exocyclic amino groups present in nucleobases and thus also changes sequence information. The method uses a mapping algorithm specifically adapted to the sequence degeneration caused by deamination events. Thus, m6A sites with partial modification levels of ∼50% were detected in defined amplicons, and this threshold can be lowered to ∼10% by combination with m6A immunoprecipitation. NOseq faithfully detected known m6A sites in human rRNA, and the long non-coding RNA MALAT1, and positively validated several m6A candidate sites, drawn from miCLIP data with an m6A antibody, in the transcriptome of Drosophila melanogaster. Conceptually related to bisulfite sequencing, NOseq presents a novel amplicon-based sequencing approach for the validation of m6A sites in defined sequences.

Genetic Alterations Precede DNA Methylation Changes in Juvenile Myelomonocytic Leukemia

Introduction Juvenile myelomonocytic leukemia (JMML) is a rare and highly aggressive myeloid malignancy of early childhood. Approximately 95% of patients have at least one mutation that leads to hyperactive RAS signaling. Both the presence of multiple mutations at diagnosis as well as altered DNA methylation are associated with a poor prognosis. Whether altered DNA methylation leads to the acquisition of additional genetic mutations or is a secondary event to genetic mutations is unknown. In this study, we analyzed a total of 33 newborn blood screening (NBS) cards from children who went on to develop JMML later in childhood. Using next generation sequencing to detect both genetic mutations and DNA methylation status we sought to determine the sequence of events that lead to the development of JMML. Patients and Methods We identified 33 patients that were born in the state of California from 1990 to 2017 who were confirmed to have JMML and were previously consented to participate in a JMML tissue bank study. Guthrie cards from these 33 patients as well as 12 healthy controls were obtained from the California Biobank Program. DNA samples (20 ng) were sequenced for mutations using a custom amplicon-based sequencing approach (Paragon Genomics, Hayward, CA) targeting 26 genes that are recurrently mutated in JMML. For DNA methylation profiling, 300ng of genomic DNA was processed for bisulfite conversion using the TrueMethyl oxBS Module (Tecan Genomics Inc., Redwood City, CA.) according to manufacturer's guidelines and next generetation sequencing (NGS) libraries were prepared by targeting 3000 CpG loci using custom probes for Targeted Methyl-Seq assay (Tecan Genomics Inc., Redwood City, CA). Sequence reads were trimmed using Cutadapt to remove adapters and methylation status called using Bismark. Samples were classified into one of three methylation subgroups: Low, Intermediate, or High using 1386 probes according to an international consensus definition. Results At diagnosis of JMML, somatic mutations were identified in 32 of 33 patients. Of the 32 patients with a somatic mutation present upon diagnosis, the same mutations were found in NBS cards in 13 (41%) patients using a mutant allele fraction (MAF) cut-off of 1%. Clonal mutations (MAF >15%) were found in 9 of 32 (28%) patients. Patients who had a somatic mutation detected at birth were significantly younger at diagnosis with a median age of 7.1 months (range 2.5-91.6 months) compared to patients who had none (19.8 months; p-value = 0.042). However, no difference was observed in overall or event-free survival for patients with or without somatic mutations at birth. Methylation profiling classified all NBS cards as having "low" DNA methylation using an international, consensus definition (Figure 1A). Of the 33 patients, ten also had DNA methylation profiling performed on their diagnostic JMML sample which were classified as low (LM), intermediate (IM) or high methylation (HM) (Figure 1B). Amongst patients with both NBS and JMML methylation data available, 5 had mutations present in the NBS card and all 5 had lower methylation (β values) in their NBS card compared to their JMML sample (mean 0.38 vs 0.29, p-value = 0.002). Discussion We identified somatic Ras pathway mutations at birth in 13/32 (41%) of newborns that developed JMML later in their childhood. We found that patients with a somatic mutation detected at birth were significantly younger at diagnosis compared to patients who had no mutations at birth. DNA methylation profiling of NBS cards from children who went on to develop JMML revealed methylation signatures that were similar to normal age-matched controls. All newborn cards were classified as low methylation at birth. Specifically, amongst patients who had genetic mutations present at birth, methylation changes that were eventually detected in JMML samples were not present in the NBS cards. Therefore, we conclude that aberrant DNA methylation is a secondary event to genetic changes in JMML. Figure 1: Panel A: Methylation profiling of JMML NBS and control NBS samples, classified according to the international JMML methylation consensus signature. Panel B: Methylation profiling of paired NBS and diagnosis samples from JMML patients. The star indicates the NBS methylation profile. Heatmaps (A and B) show the beta values of 1386 CpG loci used for methylation classification. Figure 1 Disclosures Loh: Pfizer: Other: Institutional Research Funding; Medisix Therapeutics: Membership on an entity's Board of Directors or advisory committees.

Rapid Genomic Characterization of SARS-CoV-2 by Direct Amplicon-Based Sequencing Through Comparison of MinION and Illumina iSeq100TM System.

Global human health is increasingly challenged by emerging viral threats, especially those observed over the last 20 years with coronavirus-related human diseases, such as the Severe Acute Respiratory Syndrome (SARS) and the Middle East Respiratory Syndrome (MERS). Recently, in late December 2019, a novel Betacoronavirus, SARS-CoV-2, originating from the Chinese city of Wuhan, emerged and was then identified as the causative agent of a new severe form of pneumonia, COVID-19. Real-time genome sequencing in such viral outbreaks is a key issue to confirm identification and characterization of the involved pathogen and to help establish public health measures. Here, we implemented an amplicon-based sequencing approach combined with easily deployable next-generation sequencers, the small and hand-held MinION sequencer and the latest most compact Illumina sequencer, the iSeq100TM system. Our results highlighted the great potential of the amplicon-based approach to obtain consensus genomes of SARS-CoV-2 from clinical samples in just a few hours. Both these mobile next-generation sequencers are proven to be efficient to obtain viral sequences and easy to implement, with a minimal laboratory environment requirement, providing useful opportunities in the field and in remote areas.

Uncovering the microbiome of invasive sympatric European brown hares and European rabbits in Australia.

BackgroundEuropean brown hares (Lepus europaeus) and European rabbits (Oryctolagus cuniculus) are invasive pest species in Australia, with rabbits having a substantially larger environmental impact than hares. As their spatial distribution in Australia partially overlaps, we conducted a comparative microbiome study to determine how the composition of gastrointestinal microbiota varies between these species, since this may indicate species differences in diet, physiology, and other internal and external factors.MethodsWe analysed the faecal microbiome of nine wild hares and twelve wild rabbits from a sympatric periurban reserve in Canberra, Australia, using a 16S rRNA amplicon-based sequencing approach. Additionally, we compared the concordance between results from Illumina and Nanopore sequencing platforms.ResultsWe identified significantly more variation in faecal microbiome composition between individual rabbits compared to hares, despite both species occupying a similar habitat. The faecal microbiome in both species was dominated by the phyla Firmicutes and Bacteroidetes, typical of many vertebrates. Many phyla, including Actinobacteria, Proteobacteria and Patescibacteria, were shared between rabbits and hares. In contrast, bacteria from phylum Verrucomicrobia were present only in rabbits, while phyla Lentisphaerae and Synergistetes were represented only in hares. We did not identify phylum Spirochaetes in Australian hares; this phylum was previously shown to be present at high relative abundance in European hare faecal samples. These differences in the composition of faecal microbiota may be indicative of less discriminate foraging behaviour in rabbits, which in turn may enable them to adapt quicker to new environments, and may reflect the severe environmental impacts that this species has in Australia.

Abstract A41: Ultrasensitive detection of diverse genomic alterations in hematological malignancies using a targeted amplicon-based sequencing approach

Abstract Background: Hematologic malignancies, including acute myeloid leukemia (AML), myelodysplastic syndromes (MDS), myeloproliferative neoplasms (MPN), acute lymphoblastic leukemia (ALL), and multiple myeloma (MM), are characterized by complex mutational patterns with great inter- and intrapatient diversity of mutations. Targeted detection of somatic cancer-associated variants by next-generation sequencing technology has demonstrated clinical utility in hematologic malignancies, with recurrent somatic mutations being identifiable in most AML patients and nearly 90% of MDS patients. DNA-level structural rearrangements, copy number variations (CNVs), and microsatellite instability (MSI), particularly in therapy-related AML, are other key targets with clinical relevance. Appropriate test sensitivity of mutation detection is necessary for the determination of relapse and emergence of resistance mutations. An ultrasensitive amplicon-based sequencing platform technology (AmpliMark) was applied for the detection of mutations in hematologic samples, including in DNA from peripheral blood, bone marrow, and in plasma cell-free DNA. Methods: The hematologic panel covers 45 genes (representing over 90% of mutations found in patients) and 6 microsatellite loci and utilizes an optimized bioinformatics pipeline. Analytical validation was done with reference standards and admixture samples. Clinical performance was assessed in samples from adult patients (n = 65) with diagnoses including AML, ALL, MM, and MPN, both treated and untreated. Of these, 25 samples were characterized for somatic mutations using orthogonal methods and the remaining were processed as routine clinical samples. Results: Analytical validation of the panel demonstrated lower limits of variant detection to be 0.1% for mutations, 0.5% for structural rearrangements, and 5% for MSI. A high average deduplicated depth of coverage of 9375x was achieved for DNA from bone marrow and peripheral blood. The method was able to detect all orthogonally confirmed mutations for a concordance rate of 100%. In the unselected series of patient samples, at least one somatic mutation was confirmed in 85% of cases, with TET2, DNMT3A, TP53, KIT, JAK2, RUNX1, SF3B1, ASXL1, IDH1, NPM1 mutations, FLT3 internal tandem duplication, and BCR-ABL1 fusion most frequently seen. Allele frequencies detected ranged from 0.06% to over 35%. Mutations in multiple genes were observed in 70% of cases. For 10 patients, matched bone marrow and peripheral blood samples were tested and 100% mutation match was observed in the two hematologic compartments with generally higher allele frequencies in bone marrow fraction. Conclusion: We report the application of a technology platform (Amplimark) for comprehensive genomic profiling of hematologic malignancies. Excellent concordance to standard methods and among matched samples is seen, including at low allele frequencies, suggesting clinical utility of the method for hematologic malignancies. Citation Format: Yukti Choudhury, Chae Yin Cher, Min-Han Tan, Zi Yi Lim. Ultrasensitive detection of diverse genomic alterations in hematological malignancies using a targeted amplicon-based sequencing approach [abstract]. In: Proceedings of the AACR Special Conference on Advances in Liquid Biopsies; Jan 13-16, 2020; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(11_Suppl):Abstract nr A41.

A Survey of the Potential Ectomycorrhizal Fungi Associated With Nursery Seedlings of Seven Species of Exotic Quercus in China

ABSTRACT Seven species of Quercus have been introduced into China from the United States for their ecological and economic values. The beneficial association between Quercus trees and ectomycorrhizal (ECM) fungi is well recognized. However, the ECM fungal communities associated with these exotic oak trees grown in new environments are currently unknown. The aims of this work were to investigate the mycorrhizal status of nursery seedlings and identify the fungal communities in them. All Quercus tree species examined exhibited well-developed ECM roots. However, neither the morphology nor the color of the observed ECM types was distinctive, suggesting reduced ECM fungal diversity. The combined application of isolation and amplicon-based sequencing approaches revealed that the mycorrhizal taxa represented 5 orders and 12 genera in the Basidiomycota, but only the dominant genus Scleroderma (Boletales) was detected by both approaches. In addition, Scleroderma exhibited the lowest degree of host specificity, but relatively high interspecific genetic diversity. Isolates assigned to Sistotrema displayed taxonomic novelties based on sequence similarity searches against the GenBank database. Non-metric multidimensional scaling analysis showed a degree of differentiation of ECM communities among Quercus species. Our results provide insights into the identities of exotic Quercus-associated ECM fungi and will improve symbiosis-based seedling nursing technology.

Pathogen Detection and Microbiome Analysis of Infected Wheat Using a Portable DNA Sequencer

Fungal diseases of plants are responsible for major losses in agriculture, highlighting the need for rapid and accurate identification of plant pathogens. Disease outcomes are often defined not only by the main pathogen but are influenced by diverse microbial communities known as the microbiome at sites of infection. Here we present the first use of whole genome shot-gun sequencing with a portable DNA sequencing device as a method for the detection of fungal pathogens from wheat (Triticum aestivum) in a standard molecular biology laboratory. The data revealed that our method is robust and applicable to the diagnosis of fungal diseases including wheat stripe rust (caused by Puccinia striiformis f. sp. tritici), Septoria tritici blotch (caused by Zymoseptoria tritici), and yellow leaf spot (caused by Pyrenophora tritici repentis). We also identified the bacterial genus Pseudomonas co-present with Puccinia and Zymoseptoria but not Pyrenophora infections. One limitation of the method is the over-representation of redundant wheat genome sequences from samples. This could be addressed by long-range amplicon-based sequencing approaches in future studies, which specifically target nonhost organisms. Our work outlines a new approach for detection of a broad range of plant pathogens and associated microbes using a portable sequencer in a standard laboratory, providing the basis for future development of an on-site disease monitoring system. [Formula: see text] Copyright © 2019 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .

An amplicon-based sequencing approach for the study of aeromycology.

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Application of a novel amplicon-based sequencing approach reveals the diversity of the Bacillus cereus group in stored raw and pasteurized milk

Members of the Bacillus cereus sensu lato (B. cereus group) are spore-forming organisms commonly associated with spoilage of milk and dairy products. Previous studies have shown, by using 16S marker gene sequencing, that the genus Bacillus is part of the core microbiota of raw bovine milk and that some members of this genus are able to grow during sub-optimal storage (8 °C) of pasteurized consumption milk. Here, the composition of this genus in pasteurized consumption milk samples, collected from two dairies, over a one-year period and stored at 4 or 8 °C up to the end of shelf life is uncovered. Our results show that the B. cereus group is the dominant Bacillus group in stored consumption milk. By applying a new marker gene sequencing approach, several dominating phylogenetic clusters were identified within the B. cereus group populations from the milk samples. There was a higher phylogenetic diversity among bacteria from milk stored at 8 °C compared to milk stored at 4 °C. Sampling period and the dairy the samples were collected from, also significantly influenced the diversity, which shows that the B. cereus group population in consumption milk is heterogeneous and subjected to temporal and spatial changes. The new approach applied in this study will facilitate the identification of isolates within the B. cereus group, of which some are potential spoilage bacteria and pathogenic contaminants of milk and dairy products.

A Gene Panel NGS-Based Strategy for Genomic Characterization of Acute Myeloid Leukemias (AMLs)

AMLs are clonal disorders characterized by high genomic heterogeneity and several chromosomal and molecular alterations affecting patients' outcome. In about 40% of AML patients who do not show any citogenetic alteration, sequencing analysis identified different gene mutations which play a pivotal role in leukemogenesis and have a negative prognostic impact: FLT3, ASXL1, TET2, IDH1, IDH2, RUNX1, CBL, CEBPα, DNMT3A and TP53. Conventional Sanger sequencing may detect clones representing more than 20% of the total tumor population, whereas Next Generation Sequencing (NGS) can identify mutations in less than 1% of leukemic cell burden. The detection of these variants is relevant because they can play an important role in driving drug resistance and disease relapse and for biologic risk assessment.To that purpose, we designed a 23-genes panel including: FLT3, DNMT3A, RUNX1, ASXL1, IDH1, IDH2, BCOR, NRAS, KRAS, TET2, TP53, U2AF1, ZRSR2, SF3B1, SRSF2, CBL, CEBPα, EZH2, NPM1, TERT, TERC, ETV6, GATA2. By using a 454 GS Junior by Roche Diagnostics with an amplicon-based sequencing approach and performing three sequencing runs per sample in order to reach a sensitivity of at least 1%, we settled an investigative strategy in order to assess the genomic profile of AMLs at diagnosis and possibly at the time of relapse or resistance. By this approach, we were able to confirm all the variants (i.e. FLT3, NPM1) previously documented with conventional tests; to reveal the presence of variants under the Sanger threshold of 20% in the genes resulted as wild type with routinely analysis; to evaluate the AML clonal heterogeneity, by assessing the coexistence of several mutated clones which have been described to be related to drug resistance, poor prognosis or to prior myelodysplastic syndrome.This gene panel NGS-based strategy may be proposed as a highly accurate and sensitive approach for genomic characterization of acute myeloid leukemias and as an useful tool for planning a target and personalized AML therapy. DisclosuresNo relevant conflicts of interest to declare.