Next-generation Sequencing Protocols Research Articles

Next-Generation Sequencing Approaches to Define the Role of the Autophagy Lysosomal Pathway in Human Disease: The Example of LysoPlex.

Next-Generation Sequencing (NGS) technologies have deeply changed the throughput of genetic testing allowing analyzing millions of DNA fragments in parallel. One key application is the understanding of genetically heterogeneous and complex diseases where 50-100 different genes may converge to control the same pathways. These disorders cannot be studied using traditional approaches, based on gene-by-gene Sanger sequencing. We have set up an NGS protocol based on a specific selection of DNA regions belonging to about 900 genes of the autophagy-lysosomal (ALP) pathway. We here specify all the technical steps and challenges of our protocol, named LysoPlex. This is based on the Haloplex technology and together with high-coverage sequencing empowers a high and uniform coverage of ALP genes. LysoPlex outplays other NGS applications in sensitivity and specificity, providing an accurate picture of all variations in ALP genes.

Informativeness of NGS Analysis for Vaginal Fluid Identification.

The identification of vaginal fluids in forensic examinations plays an important role in crime scene reconstruction. Molecular detection of vaginal bacterial communities can lead to the correct discrimination of body fluids. These kinds of studies can be performed through multiplex real-time PCR using primers for a specific selection of bacteria. The availability of next-generation sequencing (NGS) protocols provided for the extension of the analysis to evaluate the prokaryotes present in specimens. In this study, DNA was extracted from 18 samples (vaginal, oral, fecal, yoghurt) and analyzed by real-time PCR and NGS. The comparison between the two approaches has demonstrated that the information developed through NGS can augment the more conventional real-time PCR detection of a few key bacterial species to provide a more probative result and the correct identification of vaginal fluid from samples that are more forensically challenged.

Sequence Capture and Phylogenetic Utility of Genomic Ultraconserved Elements Obtained from Pinned Insect Specimens.

Obtaining sequence data from historical museum specimens has been a growing research interest, invigorated by next-generation sequencing methods that allow inputs of highly degraded DNA. We applied a target enrichment and next-generation sequencing protocol to generate ultraconserved elements (UCEs) from 51 large carpenter bee specimens (genus Xylocopa), representing 25 species with specimen ages ranging from 2–121 years. We measured the correlation between specimen age and DNA yield (pre- and post-library preparation DNA concentration) and several UCE sequence capture statistics (raw read count, UCE reads on target, UCE mean contig length and UCE locus count) with linear regression models. We performed piecewise regression to test for specific breakpoints in the relationship of specimen age and DNA yield and sequence capture variables. Additionally, we compared UCE data from newer and older specimens of the same species and reconstructed their phylogeny in order to confirm the validity of our data. We recovered 6–972 UCE loci from samples with pre-library DNA concentrations ranging from 0.06–9.8 ng/μL. All investigated DNA yield and sequence capture variables were significantly but only moderately negatively correlated with specimen age. Specimens of age 20 years or less had significantly higher pre- and post-library concentrations, UCE contig lengths, and locus counts compared to specimens older than 20 years. We found breakpoints in our data indicating a decrease of the initial detrimental effect of specimen age on pre- and post-library DNA concentration and UCE contig length starting around 21–39 years after preservation. Our phylogenetic results confirmed the integrity of our data, giving preliminary insights into relationships within Xylocopa. We consider the effect of additional factors not measured in this study on our age-related sequence capture results, such as DNA fragmentation and preservation method, and discuss the promise of the UCE approach for large-scale projects in insect phylogenomics using museum specimens.

A technical application of quantitative next generation sequencing for chimerism evaluation.

At present, the most common genetic diagnostic method for chimerism evaluation following hematopoietic stem cell transplantation is microsatellite analysis by capillary electrophoresis. The main objective was to establish, through repeated analysis over time, if a complete chimerism was present, or if the mixed chimerism was stable, increasing or decreasing over time. Considering the recent introduction of next generation sequencing (NGS) in clinical diagnostics, a detailed study evaluating an NGS protocol was conducted, coupled with a custom bioinformatics pipeline, for chimerism quantification. Based on the technology of Ion AmpliSeq, a 44-amplicon custom chimerism panel was designed, and a custom bioinformatics pipeline dedicated to the genotyping and quantification of NGS data was coded. The custom chimerism panel allowed identification of an average of 16 informative recipient alleles. The limit of detection of the protocol was fixed at 1% due to the NGS background (<1%). The protocol followed the standard Ion AmpliSeq library preparation and Ion Torrent Personal Genome Machine guidelines. Overall, the present study added to the scientific literature, identifying novel technical details for a possible future application of NGS for chimerism quantification.

Unbiased Deep Sequencing of RNA Viruses from Clinical Samples.

Here we outline a next-generation RNA sequencing protocol that enables de novo assemblies and intra-host variant calls of viral genomes collected from clinical and biological sources. The method is unbiased and universal; it uses random primers for cDNA synthesis and requires no prior knowledge of the viral sequence content. Before library construction, selective RNase H-based digestion is used to deplete unwanted RNA — including poly(rA) carrier and ribosomal RNA — from the viral RNA sample. Selective depletion improves both the data quality and the number of unique reads in viral RNA sequencing libraries. Moreover, a transposase-based 'tagmentation' step is used in the protocol as it reduces overall library construction time. The protocol has enabled rapid deep sequencing of over 600 Lassa and Ebola virus samples-including collections from both blood and tissue isolates-and is broadly applicable to other microbial genomics studies.

Unbiased Deep Sequencing of RNA Viruses from Clinical Samples

Here we outline a next-generation RNA sequencing protocol that enables de novo assemblies and intra-host variant calls of viral genomes collected from clinical and biological sources. The method is unbiased and universal; it uses random primers for cDNA synthesis and requires no prior knowledge of the viral sequence content. Before library construction, selective RNase H-based digestion is used to deplete unwanted RNA — including poly(rA) carrier and ribosomal RNA — from the viral RNA sample. Selective depletion improves both the data quality and the number of unique reads in viral RNA sequencing libraries. Moreover, a transposase-based 'tagmentation' step is used in the protocol as it reduces overall library construction time. The protocol has enabled rapid deep sequencing of over 600 Lassa and Ebola virus samples-including collections from both blood and tissue isolates-and is broadly applicable to other microbial genomics studies.

A Universal Next-Generation Sequencing Protocol To Generate Noninfectious Barcoded cDNA Libraries from High-Containment RNA Viruses.

Several biosafety level 3 and/or 4 (BSL-3/4) pathogens are high-consequence, single-stranded RNA viruses, and their genomes, when introduced into permissive cells, are infectious. Moreover, many of these viruses are select agents (SAs), and their genomes are also considered SAs. For this reason, cDNAs and/or their derivatives must be tested to ensure the absence of infectious virus and/or viral RNA before transfer out of the BSL-3/4 and/or SA laboratory. This tremendously limits the capacity to conduct viral genomic research, particularly the application of next-generation sequencing (NGS). Here, we present a sequence-independent method to rapidly amplify viral genomic RNA while simultaneously abolishing both viral and genomic RNA infectivity across multiple single-stranded positive-sense RNA (ssRNA+) virus families. The process generates barcoded DNA amplicons that range in length from 300 to 1,000bp, which cannot be used to rescue a virus and are stable to transport at room temperature. Our barcoding approach allows for up to 288 barcoded samples to be pooled into a single library and run across various NGS platforms without potential reconstitution of the viral genome. Our data demonstrate that this approach provides full-length genomic sequence information not only from high-titer virion preparations but it can also recover specific viral sequence from samples with limited starting material in the background of cellular RNA, and it can be used to identify pathogens from unknown samples. In summary, we describe a rapid, universal standard operating procedure that generates high-quality NGS libraries free of infectious virus and infectious viral RNA. IMPORTANCE This report establishes and validates a standard operating procedure (SOP) for select agents (SAs) and other biosafety level 3 and/or 4 (BSL-3/4) RNA viruses to rapidly generate noninfectious, barcoded cDNA amenable for next-generation sequencing (NGS). This eliminates the burden of testing all processed samples derived from high-consequence pathogens prior to transfer from high-containment laboratories to lower-containment facilities for sequencing. Our established protocol can be scaled up for high-throughput sequencing of hundreds of samples simultaneously, which can dramatically reduce the cost and effort required for NGS library construction. NGS data from this SOP can provide complete genome coverage from viral stocks and can also detect virus-specific reads from limited starting material. Our data suggest that the procedure can be implemented and easily validated by institutional biosafety committees across research laboratories.

Navigating highly homologous genes in a molecular diagnostic setting: a resource for clinical next-generation sequencing.

Next-generation sequencing (NGS) is now routinely used to interrogate large sets of genes in a diagnostic setting. Regions of high sequence homology continue to be a major challenge for short-read technologies and can lead to false-positive and false-negative diagnostic errors. At the scale of whole-exome sequencing (WES), laboratories may be limited in their knowledge of genes and regions that pose technical hurdles due to high homology. We have created an exome-wide resource that catalogs highly homologous regions that is tailored toward diagnostic applications. This resource was developed using a mappability-based approach tailored to current Sanger and NGS protocols. Gene-level and exon-level lists delineate regions that are difficult or impossible to analyze via standard NGS. These regions are ranked by degree of affectedness, annotated for medical relevance, and classified by the type of homology (within-gene, different functional gene, known pseudogene, uncharacterized noncoding region). Additionally, we provide a list of exons that cannot be analyzed by short-amplicon Sanger sequencing. This resource can help guide clinical test design, supplemental assay implementation, and results interpretation in the context of high homology.Genet Med 18 12, 1282-1289.

A Universal Next-Generation Sequencing Protocol To Generate Noninfectious Barcoded cDNA Libraries from High-Containment RNA Viruses

A Universal Next-Generation Sequencing Protocol To Generate Noninfectious Barcoded cDNA Libraries from High-Containment RNA Viruses

Adenylylation of small RNA sequencing adapters using the TS2126 RNA ligase I.

Many high-throughput small RNA next-generation sequencing protocols use 5′ preadenylylated DNA oligonucleotide adapters during cDNA library preparation. Preadenylylation of the DNA adapter's 5′ end frees from ATP-dependence the ligation of the adapter to RNA collections, thereby avoiding ATP-dependent side reactions. However, preadenylylation of the DNA adapters can be costly and difficult. The currently available method for chemical adenylylation of DNA adapters is inefficient and uses techniques not typically practiced in laboratories profiling cellular RNA expression. An alternative enzymatic method using a commercial RNA ligase was recently introduced, but this enzyme works best as a stoichiometric adenylylating reagent rather than a catalyst and can therefore prove costly when several variant adapters are needed or during scale-up or high-throughput adenylylation procedures. Here, we describe a simple, scalable, and highly efficient method for the 5′ adenylylation of DNA oligonucleotides using the thermostable RNA ligase 1 from bacteriophage TS2126. Adapters with 3′ blocking groups are adenylylated at >95% yield at catalytic enzyme-to-adapter ratios and need not be gel purified before ligation to RNA acceptors. Experimental conditions are also reported that enable DNA adapters with free 3′ ends to be 5′ adenylylated at >90% efficiency.

DNA barcodes from century-old type specimens using next-generation sequencing.

Type specimens have high scientific importance because they provide the only certain connection between the application of a Linnean name and a physical specimen. Many other individuals may have been identified as a particular species, but their linkage to the taxon concept is inferential. Because type specimens are often more than a century old and have experienced conditions unfavourable for DNA preservation, success in sequence recovery has been uncertain. This study addresses this challenge by employing next-generation sequencing (NGS) to recover sequences for the barcode region of the cytochrome c oxidase 1 gene from small amounts of template DNA. DNA quality was first screened in more than 1800 century-old type specimens of Lepidoptera by attempting to recover 164-bp and 94-bp reads via Sanger sequencing. This analysis permitted the assignment of each specimen to one of three DNA quality categories--high (164-bp sequence), medium (94-bp sequence) or low (no sequence). Ten specimens from each category were subsequently analysed via a PCR-based NGS protocol requiring very little template DNA. It recovered sequence information from all specimens with average read lengths ranging from 458 bp to 610 bp for the three DNA categories. By sequencing ten specimens in each NGS run, costs were similar to Sanger analysis. Future increases in the number of specimens processed in each run promise substantial reductions in cost, making it possible to anticipate a future where barcode sequences are available from most type specimens.

Next generation DNA sequencing of full length HLA class I genes yields concordant assignments and identifies the likelihood of variation outside of exons encoding the antigen recognition domain

Aim We have developed an allele-level resolution next generation sequencing (NGS) strategy for HLA class I alleles and used it to explore HLA diversity in a registry donor population. Methods Long-range amplification of full-length HLA-A, -B, -C loci was performed in separate polymerase chain reactions using DNA from a buccal swab. Amplicons from one individual were then pooled and sheared by sonication. A library was constructed using Illumina’s TruSeq Nano kit and the DNA fragments tagged with one unique index combination. Libraries from 96 individuals were combined and sequenced simultaneously in a single 500 cycle (V2) paired-end run using an Illumina MiSeq. Data analysis used Conexio Genomics AssignTM MPS software. Results The typing protocol was validated with 552 donor registry samples previously typed by Sanger sequencing and probe hybridization at single genotype G level resolution, yielding a concordance of 99%. NGS yielded primarily single allele-level genotypes. Excluding amplification failures and inability to obtain an NGS assignment (4% loci), 32 discrepant NGS allele assignments were attributed to poor PCR amplification or loss of reads during assembly by software. Complete exon sequences identified 53 known A alleles (48 common or well-documented (CWD)), 89 B alleles (83 CWD), 52 C alleles (47 CWD). Ten potential new alleles with exon variation outside of exons 2 + 3 and over 25% of samples with potential intron variation require further study. The ability to make assignments based on specific regions of the sequence (e.g., exons only) was critical so that typings could be rapidly transmitted to the registry prior to a more comprehensive analysis of the diversity. Conclusions The protocol for NGS of class I alleles provides accurate results and should facilitate high volume high resolution typing of donor registry populations. The extent of new variation in exons is limited but the potential for intron variation is high. C. Hurley: Other (Identify); Company/Organization; Intellectual property.

Influence of next-generation sequencing and storage conditions on miRNA patterns generated from PAXgene blood.

Whole blood derived miRNA signatures determined by Next-Generation Sequencing (NGS) offer themselves as future minimally invasive biomarkers for various human diseases. The PAXgene system is a commonly used blood storage system for miRNA analysis. Central to all miRNA analyses that aim to identify disease specific miRNA signatures, is the question of stability and variability of the miRNA profiles that are generated by NGS. We characterized the influence of five different conditions on the genome wide miRNA expression pattern of human blood isolated in PAXgene RNA tubes. In detail, we analyzed 15 miRNomes from three individuals. The blood was subjected to different numbers of freeze/thaw cycles and analyzed for the influence of storage at -80 or 8 °C. We also determined the influence of blood collection and NGS preparations on the miRNA pattern isolated from a single individual, which has been sequenced 10 times. Here, five PAXGene tubes were consecutively collected that have been split in two replicates, representing two experimental batches. All samples were analyzed by Illumina NGS. For each sample, approximately 20 million NGS reads have been generated. Hierarchical clustering and Principal Component Analysis (PCA) showed an influence of the different conditions on the miRNA patterns. The effects of the different conditions on miRNA abundance are, however, smaller than the differences that are due to interindividual variability. We also found evidence for an influence of the NGS measurement on the miRNA pattern. Specifically, hsa-miR-1271-5p and hsa-miR-182-5p showed coefficients of variation above 100% indicating a strong influence of the NGS protocol on the abundance of these miRNAs.

Characterization and comparative analysis of a simian foamy virus complete genome isolated from Brazilian capuchin monkeys

Foamy viruses infect a wide range of placental mammals, including primates. However, despite of great diversity of New World primates, only three strains of neotropical simian foamy viruses (SFV) have been described. Only after 40 years since serological characterization, the complete sequence of an SFVcap strain infecting a family of six capuchin monkeys (Sapajus xanthosternos) was obtained. Co-culture of primate peripheral blood mononuclear cells with Cf2Th canine cells was established and monitored for the appearance of cytopathic effects, PCR amplification of integrated SFV proviral genome and viral reverse transcriptase activity. The novel SFVcap was fully sequenced through a next-generation sequencing protocol. Phylogenetic analysis of the complete genome grouped SFVcap and SFVmar, both infecting primate species of the Cebidae family with a genetic similarity of approximately 85%. Similar ORF sizes were observed among SFV from neotropical primates, and env and pol genes were the most conserved. Neotropical SFV presented the smallest LTRs among exogenous mammalians. The novel SFVcap strain provides a valuable research tool for the FV community.

Targeted NGS meets expert clinical characterization: Efficient diagnosis of spastic paraplegia type 11

Next generation sequencing (NGS) is transforming the diagnostic approach for neurological disorders, since it allows simultaneous analysis of hundreds of genes, even based on just a broad, syndromic patient categorization. However, such an approach bears a high risk of incidental and uncertain genetic findings. We report a patient with spastic paraplegia whose comprehensive neurological and imaging examination raised a high clinical suspicion of SPG11. Thus, although our NGS pipeline for this group of disorders includes gene panel and exome sequencing, in this sample only the spatacsin gene region was captured and subsequently searched for mutations. Two probably pathogenic variants were quickly and clearly identified, confirming the diagnosis of SPG11. This case illustrates how combination of expert clinical characterization with highly oriented NGS protocols leads to a fast, cost-efficient diagnosis, minimizing the risk of findings with unclear significance.

A phase II study of afatinib in patients (pts) with metastatic human epidermal growth factor receptor (HER2)-positive trastuzumab refractory esophagogastric (EG) cancer.

59 Background: Trastuzumab combined with chemotherapy is the standard of care for pts with HER2+ EG cancer. Resistance to trastuzumab clearly emerges in this population. Afatinib, a potent ErbB Family Blocker, induced tumor regression in MSKCC HER2+ patient derived xenografts (PDX). This study assesses safety and preliminary efficacy of afatinib in patients with trastuzumab refractory EG cancer. Methods: Pts with HER2+ (IHC 3+ or FISH&gt;2.0) EG adenocarcinoma, after progression on trastuzumab, received oral afatinib 40 mg daily. Archival pre-trastuzumab tissue, tumor biopsy after progression on trastuzumab and after 1 week on afatinib is mandated on protocol for next generation sequencing (NGS), proteomics and establishment of PDX. The primary endpoint-overall clinical benefit at 4 months: stable disease (SD) or partial response (PR). Results: 20 pts treated with afatinib; median age 61, KPS 80; median 2 (1 to 4) prior trastuzumab regimens, 67% of tumors IHC3+; 33% IHC2+/FISH&gt;2.0. Common adverse events included: rash or dry skin (Grade 1/2:80%), diarrhea (Grade 1/2:60%), nausea/vomiting (grade 1/2:40%) fatigue (grade1/2: 25%). To date, 19 pts evaluable for response, 2 PRs and 6 SD, 42% disease stabilization rate (PR+SD) at 4months (4 to 13 mos). PDXs established from biopsies of 7 pts. EGFR amplification was detected in the tumor of 2 pts with PRs and 1 of 6 pts with SD. Recurrent PIK3CA, ERBB3 and MTORmutations were observed. Conclusions: Afatinib shows clinical efficacy and is well tolerated in patients with trastuzumab refractory, heavily pretreated EG cancer. Enrollment has now begun in an afatinib + trastuzumab cohort. Efforts to elucidate the mechanisms of trastuzumab resistance including validation of potential drivers of trastuzumab resistance using HER2+ PDXs are ongoing. Updated molecular and clinical data will be presented. Clinical trial information: NCT01522768.

Evaluation and application of the strand-specific protocol for next-generation sequencing.

Next-generation sequencing (NGS) has become a powerful sequencing tool, applied in a wide range of biological studies. However, the traditional sample preparation protocol for NGS is non-strand-specific (NSS), leading to biased estimates of expression for transcripts overlapped at the antisense strand. Strand-specific (SS) protocols have recently been developed. In this study, we prepared the same RNA sample by using the SS and NSS protocols, followed by sequencing with Illumina HiSeq platform. Using real-time quantitative PCR as a standard, we first proved that the SS protocol more precisely estimates gene expressions compared with the NSS protocol, particularly for those overlapped at the antisense strand. In addition, we also showed that the sequence reads from the SS protocol are comparable with those from conventional NSS protocols in many aspects. Finally, we also mapped a fraction of sequence reads back to the antisense strand of the known genes, originally without annotated genes located. Using sequence assembly and PCR validation, we succeeded in identifying and characterizing the novel antisense genes. Our results show that the SS protocol performs more accurately than the traditional NSS protocol and can be applied in future studies.

Choice of next-generation sequencing pipelines.

The next-generation sequencing (NGS) technologies are revolutionary tools which have made possible achieving remarkable advances in genetics since the beginning of the twenty-first century. Thanks to the possibility to produce large amount of sequence data, these tools are going to completely substitute other high-throughput technologies. Moreover, the large applications of NGS protocols are increasing the genetic decoding of biological systems through studies of genome anatomy and gene mapping, coupled to the transcriptome pictures. The application of NGS pipelines such as (1) de-novo genomic sequencing by mate-paired and whole-genome shotgun strategies; (2) specific gene sequencing on large bacterial communities; and (3) RNA-seq methods including whole transcriptome sequencing and Serial Analysis of Gene Expression (Sage-analysis) are fundamental in the genome-wide fields like metagenomics. Recently, the availability of these advanced protocols has allowed to overcome the usual sequencing technical issues related to the mapping specificity over standard shotgun library sequencing, the detection of large structural genomes variations and bridging sequencing gaps, as well as more precise gene annotation. In this chapter we will discuss how to manage a successful NGS pipeline from the planning of sequencing projects through the choice of the platforms up to the data analysis management.

In Chronic Myeloid Leukemia Patients on 2nd-Line Tyrosine Kinase Inhibitor Therapy, Deep Sequencing at the Time of Warning May Allow Sensitive Detection of Emerging BCR-ABL1 Mutants

▪Background and Aims: Next generation amplicon-based deep sequencing (DS) on the Roche, Illumina or Ion Torrent instruments is becoming accessible to a wider and wider number of diagnostic laboratories. Although conventional sequencing is still the gold standard, DS has been hailed by many as the future of diagnostic BCR-ABL1 kinase domain (KD) mutation screening. BCR-ABL1 KD mutations are infrequent in newly diagnosed chronic myeloid leukemia (CML) patients (pts) receiving 1st-line TKI therapy, but remain a challenge in relapsed pts, who usually display a greater genetic instability. Indeed, pts already harboring BCR-ABL1 KD mutations have a higher likelihood of developing additional, dasatinib (DAS)- or nilotinib (NIL)-resistant mutations – which is defined as a 'failure’ by the 2013 European LeukemiaNet (ELN) recommendations. Taking advantage of a next-generation amplicon sequencing design and protocol set up and validated in the framework of the IRON-II international study, we aimed to assess whether DS may allow a larger window of detection of emerging BCR-ABL1 KD mutants predicting for an impending relapse.Methods: among the imatinib (IM)-resistant CML pts who switched to 2nd-line TKI therapy and were referred to our laboratory for routine BCR-ABL1 transcript level monitoring and KD mutation screening by conventional sequencing, 51 acquired DAS- or NIL-resistant mutations after a median of 9 months (range, 3-27 months) of therapy and had leftover cDNA available at previous timepoints. To reconstruct the dynamics of mutation emergence, resequencing on a Roche GS Junior instrument was performed from the time of failure and mutation detection by conventional sequencing backwards. Runs were designed to achieve high sequencing depth, allowing reliable detection of variants down to 1% abundance. BCR-ABL1/ABL1%IS transcript levels and/or cytogenetic response, whichever available, were used to define whether the patient had an 'optimal response’, 'warning’ or 'failure’ at the time of first mutation detection by DS.Results: baseline mutation status, as assessed by conventional sequencing, was available for all the 51 CML pts included in this retrospective study; 29/51 pts were positive for BCR-ABL1 KD mutations, with switch to NIL or DAS selected accordingly. Twenty-six pts were later found to have acquired DAS-resistant mutations (T315I, n=13; F317L/V, n=10; V299L, n=3) and 25 pts were later found to have acquired NIL-resistant mutations (T315I, n=4; F359V/I/C, n=7; Y253H, n=6; E255K, n=9; one patient acquired two mutations). DS was able to backtrack the DAS- or NIL-resistant mutations to the previous sample(s) in 23/51 (45%) pts. Median mutation burden at the time of first detection by DS was 5% (range, 1-17%); median interval between detection by DS and detection by conventional sequencing was 3 months (range, 3-9 months). In 5 cases, the mutations were traceable at baseline; in the remaining cases, correlation with response at the time mutations were first detected by DS revealed a 'warning’ according to the 2013 ELN definitions of response to 2nd-line therapy in 13 cases; an 'optimal response’ in one case; a 'failure’ in 4 cases. As a control, we used DS to explore BCR-ABL1 KD mutation status in 10 randomly selected pts with 'warning’ at various timepoints, that later turned into optimal responses; no DAS- or NIL-resistant mutations were detected.Conclusions: the 2011 ELN recommendations for mutation analysis suggest BCR-ABL1 KD to be screened by conventional sequencing in case of 'failure’ of 2nd-line TKI therapy – according to the provisional definitions available at the time. Earlier detection of emerging BCR-ABL1 KD mutations allows a greater leeway in tackling drug resistance and enhancing therapeutic efficacy. Data presented herein indicate that:1) DS may reliably pick TKI-resistant mutations earlier than conventional sequencing in a proportion of pts, and that2) the recently introduced definitions of ‘warning’ may provide a rational trigger, besides ‘failure’, for DS-based BCR-ABL1 KD mutation screening in CML pts on 2nd-line TKI therapy.A prospective cost-benefits evaluation of using DS in this and other settings is warranted, and will contribute useful information to the revision of the ELN recommendations for BCR-ABL1 KD mutation analysis.Supported by: European LeukemiaNet, AIL, AIRC, FP7 NGS-PTL project, Progetto Regione-Università 2010-12 (L. Bolondi). DisclosuresSoverini:Novartis: Consultancy; Bristol-Meyers Squibb: Consultancy; Ariad: Consultancy. Castagnetti:Novartis Farma: Consultancy, Honoraria; Bristol Myers Squibb: Consultancy, Honoraria; Pfizer: Consultancy. Gugliotta:Novartis: Consultancy, Honoraria; Bristol Myers Squibb: Consultancy, Honoraria. Bonifacio:Amgen Inc.: Consultancy. Rosti:Novartis: Consultancy; Bristol-Myers Squibb: Consultancy. Baccarani:Novartis: Consultancy; Bristol-Myers Squibb: Consultancy; Ariad: Consultancy; Pfizer: Consultancy. Martinelli:NOVARTIS: Consultancy, Speakers Bureau; BMS: Consultancy, Speakers Bureau; PFIZER: Consultancy; ARIAD: Consultancy.

Microsatellites of the self-compatible nemertean Prosorhochmus americanus (Hoplonemertea)

Thousands of microsatellite loci were identified in the nemertean Prosorhochmus americanus through enrichment-isolation and next-generation sequencing protocols. We tested 48 loci for amplification and subsequently genotyped 17 loci in 30 individuals from three North American populations. Most loci were polymorphic within and across populations; six were monomorphic. The number of alleles per locus ranged from 1 to 9 (mean 4.35, SD 2.37). Cross-species amplification in four nemertean species revealed extended marker utility. These loci will enable investigations of colonization potential and natural history in this selfing, putatively introduced species.