Next-generation Sequencing Strategies Research Articles

Next-generation sequencing yields the first complete mitochondrial genome of the ruby dragonet Synchiropus sycorax (Syngnathiformes, Callionymidae)

The complete mitogenome sequence of the ruby dragonet Synchiropus sycorax was first determined using next-generation sequencing strategy in this study. The circle genome was 16,656 bp in length and consisted of 13 protein-coding genes, 2 ribosomal RNA genes, 22 transfer RNA genes, and 1 control region. The mitochondrial gene arrangement of S. sycorax is similar to those of most other fish species. Results from neighbor-joining phylogenetic analysis showed that S. sycorax clustered with S. splendidus and other species of the family Callionymidae. This study will be valuable for phylogenetic analysis of the genus Synchiropus and the other genera of the order Syngnathiformes.

Utility of Genomic Testing after Renal Biopsy

Background: Renal biopsy is the mainstay of renal pathological diagnosis. Despite sophisticated diagnostic techniques, it is not always possible to make a precise pathological diagnosis. Our aim was to identify a genetic cause of disease in patients who had undergone renal biopsy and determine if genetic testing altered diagnosis or treatment. Methods: Patients with suspected familial kidney disease underwent a variety of next-generation sequencing (NGS) strategies. The subset of these patients who had also undergone native kidney biopsy was identified. Histological specimens were reviewed by a consultant pathologist, and genetic and pathological diagnoses were compared. Results: Seventy-five patients in 47 families underwent genetic sequencing and renal biopsy. Patients were grouped into 5 diagnostic categories based on pathological diagnosis: tubulointerstitial kidney disease (TIKD; n = 18); glomerulonephritis (GN; n = 15); focal segmental glomerulosclerosis and Alport Syndrome (n = 11); thrombotic microangiopathy (TMA; n = 17); and nonspecific pathological changes (n = 14). Thirty-nine patients (52%) in 21 families (45%) received a genetic diagnosis; 13 cases (72%) with TIKD, 4 (27%) with GN, 6 (55%) with focal segmental glomerulosclerosis/Alport syndrome, and 10 (59%) with TMA and 6 cases (43%) with nonspecific features. Genetic testing resulted in changes in understanding of disease mechanism in 21 individuals (54%) in 12 families (57%). Treatment would have been altered in at least 26% of cases (10/39). Conclusions: An accurate genetic diagnosis can result in changes in clinical diagnosis, understanding of pathological mechanism, and treatment. NGS should be considered as a complementary diagnostic technique to kidney biopsy in the evaluation of patients with kidney disease.

Economic Impact of Next-Generation Sequencing Versus Single-Gene Testing to Detect Genomic Alterations in Metastatic Non-Small-Cell Lung Cancer Using a Decision Analytic Model.

The aim of the current study was to assess the economic impact of using next-generation sequencing (NGS) versus single-gene testing strategies among patients with metastatic non-small-cell lung cancer (mNSCLC) from the perspective of the Centers for Medicare & Medicaid Services (CMS) and US commercial payers. A decision analytic model considered patients who were newly diagnosed with mNSCLC who received programmed death ligand 1 and genomic alteration tests-EGFR, ALK, ROS1, BRAF, MET, HER2, RET, and NTRK1-using upfront NGS (all alterations tested simultaneously plus KRAS), sequential testing (sequence of single-gene tests), exclusionary testing (KRAS plus sequential testing), and hotspot panels (EGFR, ALK, ROS1, and BRAF tested simultaneously plus single-gene tests or NGS for MET, HER2, RET, and NTRK1). Model outcomes for each strategy were time-to-test results, the proportion of patients identified harboring alterations with or without US Food and Drug Administration-approved therapies, and total testing costs. A budget impact analysis assessed the economic effects of increasing the proportion of NGS-tested patients. In a hypothetical 1,000,000-member health plan, 2,066 Medicare-insured patients and 156 commercially insured patients were estimated to have mNSCLC and to be eligible for testing. Time-to-test results were 2.0 weeks for NGS and the hotspot panel, faster than exclusionary and sequential testing by 2.7 and 2.8 weeks, respectively. NGS was associated with cost savings for both CMS ($1,393,678; $1,530,869; and $2,140,795 less than exclusionary, sequential testing, and hotspot panels, respectively) and commercial payers ($3,809; $127,402; and $250,842 less than exclusionary, sequential testing, and hotspot panels, respectively). Increasing the proportion of NGS-tested patients translated into substantial cost savings for both CMS and commercial payers. Use of upfront NGS testing in patients with mNSCLC was associated with substantial cost savings and shorter time-to-test results for both CMS and commercial payers.

A New Next-Generation Sequencing Strategy for the Simultaneous Analysis of Mutations and Chromosomal Rearrangements at DNA Level in Acute Myeloid Leukemia Patients

Acute myeloid leukemias (AMLs) are currently genomically characterized by karyotype, fluorescence in situ hybridization (FISH), real-time quantitative PCR, and DNA sequencing. Next-generation sequencing offers the promise of detecting all genomic lesions in a single run. However, technical limitations have hampered the detection of chromosomal rearrangements, so most studies are limited to somatic mutation assessment or require the use of RNA-based strategies. To overcome these limitations, we designed a targeted-DNA capture next-generation sequencing approach associated with easy-to-perform public bioinformatic tools for one-step identification of translocations, inversions, and somatic mutations in AML. Thirty well-characterized newly diagnosed myeloid leukemia patients (27 AML and 3 chronic myeloid leukemia) were tested with the panel. Twenty-three of 24 known rearrangements, as well as one novel fusion gene that could not be detected by karyotype/fluorescence in situ hybridization/real-time quantitative PCR, were detected. This strategy also identified all chromosomal breakpoints as potential targets for future high-sensitive minimal residual disease studies. In addition, mutation analysis revealed the presence of missense protein-coding alterations in at least 1 of the 32 genes evaluated in 21 of 30 patients (70%). This strategy may represent a time- and cost-effective diagnostic method for molecular characterization in AML.

Update on pathogenesis and genetics

Despite familial aggregation had long been recognized as a common feature in Parkinson Disease (PD), only in the past twenty years the contribution of genetics has been deeply explored, with the identification of few genes clearly responsible for mendelian forms of the disease, either with autosomal dominant (SNCA, LRRK2) or recessive (PARK2/Parkin, PINK1, DJ-1, ATP13A2) inheritance. Besides these, some other genes have been found mutated in rare families, and their actual contribution remains to be confirmed. Monogenic forms only explain a small minority of PD, leaving a large proportion of cases unaccounted. It is conceivable that in the vast majority of these patients, the disease underlies a multifactorial inheritance, with several common and rare genetic variants interplaying with epigenetic and with environmental factors to reach a threshold of disease. The most relevant example are heterozygous variants of the GBA gene, which represent a strong predisposing factor for PD development, with an average relative risk of about 5; moreover, several common variants (most residing within the same genes mutated in mendelian forms of PD) were identified as susceptibility factors by large whole genome association (GWA) studies. In line with this hypothesis, the advent of next generation sequencing (NGS) technologies, which have impressively enhanced the identification of novel causative genes in most genetic disorders, has not brought the expected revolution in the field of PD genetics. Indeed, only few rarely mutated genes have been found in isolated families, and whole exome sequencing efforts in large cohorts of PD patients have often failed to identify robust novel PD candidates. While it can be safely expected that the huge amount of genetic data generated through NGS and GWA strategies contains a wealth of new relevant information to better understand the complex genetic basis of PD, the meaningful analysis of such data to extrapolate novel PD candidate genes represents a truly challenging task. A possible lead to get oriented in the maze of common and rare genetic variants emerging from genome-wide studies may derive from the analysis of pathogenic mechanisms of neurodegeneration. Intriguingly, it is clearly emerging that neuronal death results as a consequence of the derangement of very few essential and highly interconnected cellular pathways, and that many if not all genetic factors implicated in PD pathogenesis converge on, and affect, the same cellular processes, including mitochondrial dysfunction, misfolded protein damage, impairment of clearance systems, abnormal calcium handling and enhanced pro-inflammatory responses.

Prognostic implications of MRD assessment in multiple myeloma patients: comparison of Next-Generation Sequencing and Next-Generation Flow

Despite the deeper level of responses that new agents have brought to myeloma patients, some of them will eventually relapse due to the presence of resistant cells (minimal residual disease, MRD). Detecting MRD-positive cases allows the identification of patients with an increased risk of relapse, progression or death. In this context, Next-Generation Flow (NGF) and Next-Generation Sequencing (NGS) have emerged as potential tools to evaluate MRD. Because of that, we wanted to validate a commercial NGS strategy (LymphoTrack®), comparing it with NGF in the context of the Spanish Myeloma Group GEM2012MENOS65 clinical trial (ClinicalTrials.gov access number: NCT01916252).

Genetic diversity of Koala retrovirus env gene subtypes: insights into northern and southern koala populations

Koala retrovirus (KoRV) is a recently endogenized retrovirus associated with neoplasia and immunosuppression in koala populations. The virus is known to display sequence variability and to be present at varying prevalence in different populations, with animals in southern Australia displaying lower prevalence and viral loads than northern animals. This study used a PCR and next-generation sequencing strategy to examine the diversity of the KoRV env gene in both proviral DNA and viral RNA forms in two distinct populations representative of the 'northern' and 'southern' koala genotypes. The current study demonstrated that the full range of KoRV subtypes is present across both populations, and in both healthy and sick animals. KoRV-A was the predominant proviral subtype in both populations, but there was marked diversity of DNA and RNA subtypes within individuals. Many of the northern animals displayed a higher RNA viral diversity than evident in their proviral DNA, indicating relatively higher replication efficiency of non-KoRV-A subtypes. The southern animals displayed a lower absolute copy number of KoRV than the northern animals as reported previously and a higher preponderance of KoRV-A in individual animals. These discrepancies in viral replication and diversity remain unexplained but may indicate relative protection of the southern population from KoRV replication due to either viral or host factors and may represent an important protective effect for the host in KoRV's ongoing entry into the koala genome.

BRCA1/2 somatic mutation detection in formalin-fixed paraffin embedded tissue by next-generation sequencing in Korean ovarian cancer patients

IntroductionThe detection of BRCA1/2 mutations is important because PARP1 inhibitors are approved for germline and/or somatic BRCA-mutated advanced ovarian cancer. Next-generation sequencing (NGS) is increasingly used in clinical practice for BRCA1/2 mutations. The purpose of this study was to consider several conditions of NGS BRCA1/2 assay applicable to clinical laboratory tests, in particular for using formalin fixed paraffin embedded (FFPE) ovarian tissues. Materials and methodsWe selected 64 ovarian cancer patients and performed Oncomine™ BRCA assay using FFPE tissue. Effect of FFPE sample quality was analyzed by NGS quality parameters including deamination metric. Somatic variants were selected by removing germline variants of peripheral blood and interpreted as pathogenic, variants of unknown significance, and false positive. ResultsWe found a positive relationship between the number of variants over the deamination metric and FFPE age (P < 0.001) with a cutoff values of approximately 0.7 and 60 months, respectively. When comparing NGS results with Sanger sequencing, NGS misreported 3 of 15 variants using default parameters which were corrected after changing parameters. We detected somatic variants in eight patients and classified them into pathogenic (n = 3), VUS (n = 3) and false positive (n = 2). ConclusionsThis study is important for improving BRCA1/2 mutation detection capabilities of NGS analytical pipelines and strategy to overcome their limitations using FFPE tissue in ovarian cancer patients.

Variations in Oral Microbiota Composition Are Associated With a Risk of Throat Cancer.

In this study, a next-generation sequencing strategy on 16S ribosomal RNA (16S rRNA) gene was employed to analyze 70 oral samples from 32 patients with throat cancer, nine patients with vocal cord polyp, and 29 healthy individuals (normal controls). Using this strategy, we demonstrated, for the first time, that the salivary microbiota of cancer patients were significantly different from those of patients with a polyp and healthy individuals. We observed that the beta diversity of the cancer group was divergent from both the normal and polyp groups, while alpha-diversity indices such as the Chao1 estimator (P = 8.1e-05), Simpson (P = 0.0045), and Shannon (P = 0.0071) were significantly reduced in cancer patients compared with patients containing a polyp and normal healthy individuals. Linear discriminant analysis (LDA) and Kruskal–Wallis test analyses and real-time quantitative polymerase chain reaction (qPCR) verification test revealed that the genera Aggregatibacter, Pseudomonas, Bacteroides, and Ruminiclostridium were significantly enriched in the throat cancer group compared with the vocal cord polyp and normal control groups (score value >2). Finally, diagnostic models based on putatively important constituent bacteria were constructed with 87.5% accuracy [area under the curve (AUC) = 0.875, 95% confidence interval (CI): 0.695–1]. In summary, in this study we characterized, for the first time, the oral microbiota of throat cancer patients without smoking history. We speculate that these results will help in the pathogenic mechanism and early diagnosis of throat cancer.

Characterization of a cryptic KMT2A/AFF1 gene fusion by mate-pair sequencing (MPseq) in a young adult with newly diagnosed B-lymphoblastic leukemia

The detection of recurrent chromosomal rearrangements in B-lymphoblastic leukemia/lymphoma (B-ALL/LBL) is critical for patient management decisions. We present a newly diagnosed case of B-ALL in a young adult with a cryptic KMT2A/AFF1 fusion that was unappreciable by conventional chromosome and fluorescence in situ hybridization (FISH) KMT2A break-apart probe studies. To further characterize this abnormality, a next-generation sequencing strategy, mate-pair sequencing (MPseq) was performed and characterized a cryptic, insertional rearrangement that created KMT2A/AFF1 gene fusion. This case highlights the superior precision and resolution capabilities of NGS when compared to traditional cytogenetic methodologies, including conventional chromosome and FISH studies.

Economics of Genetic Testing for Diabetes.

Monogenic diabetes is an uncommon but important form of diabetes, with the most common causes benefitting from management that accounts for the genetic mutation. This often results in decreased costs and treatment burden for affected individuals. Misdiagnosis as type 1 and type 2 diabetes is common. Given the significant burden of diabetes costs to the healthcare system, it is important to assess the economic impact of incorporating genetic testing for monogenic diabetes into clinical care through formal cost-effectiveness analyses (CEAs). This article briefly summarizes the barriers to timely monogenic diabetes diagnosis and then summarizes findings from CEAs on genetic testing for monogenic diabetes. CEAs have shown that routine genetic testing of all patients with a clinical diagnosis of type 1 diabetes can be cost-saving when applied to the scenarios of neonatal diabetes or in a pediatric population. Routine screening has not been shown to be cost-effective in adult populations. However, next-generation sequencing strategies and applying biomarkers to identify and limit genetic testing to people most likely to have monogenic diabetes are promising ways to make testing strategies cost-effective. CEAs have shown that genetic testing for monogenic diabetes diagnosis can be cost-effective or cost-saving and should guide insurers to consider broader coverage of these tests, which would lead to accurate and timely diagnosis and impact treatment and clinical outcomes.

MitoMatters - Targeted next generation sequencing identifies novel pathogenic variants and provides molecular diagnoses in a cohort of pediatric and adult patients with unexplained mitochondrial dysfunction

Mitochondrial diseases (MD) are a group of rare inherited disorders, characterized by phenotypic heterogeneity, with hitherto no effective therapeutic options. The aim of this study was to develop a next generation sequencing (NGS) strategy, by using a custom gene panel and whole mitochondrial genome, to identify the disease causing pathogenic variants in 146 patients suspicious of MD. The molecular analysis of this cohort revealed six novel and 15 described pathogenic variants, as well as 26 variants of unknown significance. Our findings are expanding the mutational landscape of these disorders and support the use of a NGS strategy for a higher diagnostic yield.

Characterization of a t(1;2)(p36;p21) involving the PRDM16 gene region by mate-pair sequencing (MPseq) in a patient with newly diagnosed acute myeloid leukemia with myelodysplasia-related changes

Per the 2017 WHO, several translocations have been described that are sufficient for the diagnosis of acute myeloid leukemia with myelodysplasia-related changes (AML-MRC) (assuming no prior therapy and ≥ 20% myeloblasts present in blood or bone marrow), including the t(1;3)(p36;q21). This translocation juxtaposes the RPN1 gene (3q21.2) promoter upstream of the PRDM16 gene (1p36) resulting in PRDM16 overexpression. While uncommon, PRDM16 overexpression is considered an unfavorable prognostic finding in myeloid neoplasms. A variant PRDM16 rearrangement t(1;2)(p36;p21) has been rarely described in various hematologic neoplasms, including two cases of myelodysplastic syndrome and one case each of myelofibrosis and T-lymphoblastic leukemia. We describe the first case to our knowledge of t(1;2)(p36;p21) observed in AML-MRC. In addition, a next-generation sequencing strategy, mate-pair sequencing (MPseq) was performed and revealed the promoter 2 region of THADA (2p21) was juxtaposed upstream from PRDM16 which may be responsible for PRDM16 overexpression that has been reported in hematologic neoplasms harboring the t(1;2)(p36;p21).

Optimization of Next-Generation Sequencing Technologies for von Hippel Lindau (VHL) Mosaic Mutation Detection and Development of Confirmation Methods

Von Hippel-Lindau disease (VHL) is a monogenic disorder characterized by the development of tumors affecting the central nervous system, kidney, pancreas, or adrenal glands, and due to germline mutations in the VHL tumor suppressor gene. About 5% of patients with a typical VHL phenotype have no mutation detected by conventional techniques, so a postzygotic VHL mosaicism can be suspected. The aim of this study was therefore to implement a next-generation sequencing (NGS) strategy for VHL mosaic mutation detection, including an optimization of the original Personal Genome Machine design by enrichment with oligonucleotides corresponding to amplicons with insufficient depth of coverage. Two complementary strategies were developed for the confirmation of mosaic mutations identified by NGS, SNaPshot for variants present at an allelic ratio greater than 5%, and droplet digital PCR for allelic ratio above 1%. VHL mutant plasmids were generated to assess VHL mosaic mutation detection in different exons and to set up an internal quality control that could be included in each run or regularly to validate the assay. This strategy was applied to 47 patients with a suggestive or clinical VHL disease, and mosaic mutations were identified in 8.5% of patients. In conclusion, NGS technologies combined with SNaPshot or droplet digital PCR allow the detection and confirmation of mosaic mutations in a clinical laboratory setting.

Complete Endogenous Retrovirus Genome Sequence from a Brazilian Vampire Bat (Desmodus rotundus).

The strain Desmodus rotundus endogenous retrovirus (DrERV) QR09 was obtained from a bat tissue sample collected from Desmodus rotundus in the Brazilian rain forest. The complete genome was sequenced using the next-generation sequencing strategy. The full-length genome of DrERV QR09 is 8,256 nucleotides in length and showed high similarity with other DrERVs.

Genetic Mutation Profiles in Korean Patients with Inherited Retinal Diseases.

BackgroundBecause of genetically and phenotypically heterogenous features, identification of causative genes for inherited retinal diseases (IRD) is essential for diagnosis and treatment in coming gene therapy era. To date, there are no large-scale data of the genes responsible for IRD in Korea. The aim of this study was to identify the distribution of genetic defects in IRD patients in Korea.MethodsMedical records and DNA samples from 86 clinically diagnosed IRD patients were consecutively collected between July 2011 and May 2015. We applied the next-generation sequencing strategy (gene panel) for screening 204 known pathogenic genes associated with IRD.ResultsMolecular diagnoses were made in 38/86 (44.2%) IRD patients: 18/44 (40.9%) retinitis pigmentosa (RP), 8/22 (36.4%) cone dystrophy, 6/7 (85.7%) Stargardt disease, 1/1 (100%) Best disease, 1/1 (100%) Bardet-Biedl syndrome, 1/1 (100%) congenital stationary night blindness, 1/1 (100%) choroideremia, and 2/8 (25%) other macular dystrophies. ABCA4 was the most common causative gene associated with IRD and was responsible for causing Stargardt disease (n = 6), RP (n = 1), and cone dystrophy (n = 1). In particular, mutations in EYS were found in 4 of 14 autosomal recessive RP (29%). All cases of Stargardt disease had a mutation in the ABCA4 gene with an autosomal recessive trait.ConclusionThis study provided the distribution of genetic mutations responsible for causing IRD in the Korean patients. This data will serve as a reference for future genetic screening and treatment for Korean IRD patients.

The oculoauriculofrontonasal syndrome: Further clinical characterization and additional evidence suggesting a nontraditional mode of inheritance.

The oculoauriculofrontonasal syndrome (OAFNS) is a rare disorder characterized by the association of frontonasal dysplasia (widely spaced eyes, facial cleft, and nose abnormalities) and oculo-auriculo-vertebral spectrum (OAVS)-associated features, such as preauricular ear tags, ear dysplasia, mandibular asymmetry, epibulbar dermoids, eyelid coloboma, and costovertebral anomalies. The etiology is unknown so far. This work aimed to identify molecular bases for the OAFNS. Among a cohort of 130 patients with frontonasal dysplasia, accurate phenotyping identified 18 individuals with OAFNS. We describe their clinical spectrum, including the report of new features (micro/anophtalmia, cataract, thyroid agenesis, polymicrogyria, olfactory bulb hypoplasia, and mandibular cleft), and emphasize the high frequency of nasal polyps in OAFNS (56%). We report the negative results of ALX1, ALX3, and ALX4 genes sequencing and next-generation sequencing strategy performed on blood-derived DNA from respectively, four and four individuals. Exome sequencing was performed in four individuals, genome sequencing in one patient with negative exome sequencing result. Based on the data from this series and the literature, diverse hypotheses can be raised regarding the etiology of OAFNS: mosaic mutation, epigenetic anomaly, oligogenism, or nongenetic cause. In conclusion, this series represents further clinical delineation work of the rare OAFNS, and paves the way toward the identification of the causing mechanism.

Diversity of genetic events associated with MLH1 promoter methylation in Lynch syndrome families with heritable constitutional epimutation

PurposeConstitutional epimutations are an alternative to genetic mutations in the etiology of genetic diseases. Some of these epimutations, termed secondary, correspond to the epigenetic effects of cis-acting genetic defects transmitted to the offspring following a Mendelian inheritance pattern. In Lynch syndrome, a few families with such apparently heritable MLH1 epimutations have been reported so far. MethodsWe designed a long-range polymerase chain reaction next-generation sequencing strategy to screen MLH1 entire gene and applied it to 4 French families with heritable epimutations and 10 additional patients with no proven transmission of their epimutations. ResultsThis strategy successfully detected the insertion of an Alu element in MLH1 coding sequence in one family. Two previously unreported MLH1 variants were also identified in other epimutation carriers: a nucleotide substitution within intron 1 and a single-nucleotide deletion in the 5′-UTR. Detection of a partial MLH1 duplication in another family required multiplex ligation-dependent probe amplification technology. We demonstrated the segregation of these variants with MLH1 methylation and studied the functional consequences of these defects on transcription. ConclusionThis is the largest cohort of patients with MLH1 secondary epimutations associated with a broad spectrum of genetic defects. This study provides further insight into the complexity of molecular mechanisms leading to secondary epimutations.

Genetic Drivers of Pancreatic Cancer Are Identical Between the Primary Tumor and a Secondary Lesion in a Long-Term (>5 Years) Survivor After a Whipple Procedure.

Background: A new mass in the remnant pancreas of a patient with previously resected pancreatic ductal adenocarcinoma (PDA) typically represents either a recurrence of the initial primary tumor or a second primary tumor. Recent advances in next-generation sequencing (NGS) strategies allow us to compare the genetic makeup of primary and secondary lesions.Case presentation: A 50-year-old Caucasian female presented for a surgical evaluation of a new biopsy-proven PDA at the junction of the body and tail of the pancreas. Six years prior, in 2011, the patient was found to have a T3N0M0 PDA of the pancreatic head, which was surgically resected with a classic Whipple procedure and concurrent hemicolectomy. Pathology showed pancreatic intraepithelial neoplasia grade 2 and PDA with negative surgical margins, positive perineural spread, and negative lymphovascular spread, and the patient received adjuvant chemotherapy and local radiation. In 2017, she was diagnosed with a new PDA lesion in the remaining pancreatic body far from the previous anastomosis site and was taken to surgery for a completion pancreatectomy and revision of the gastrojejunostomy. NGS was performed on both specimens. Both lesions shared identical mutations in KRAS, TP53, and CDKN2A genes. Amplifications of MYC and mutant KRAS were identified in the 2017 tumor and an ACVR1B mutation was identified in the 2011 tumor, but was not found in the 2017 tumor.Conclusions: This case demonstrates the ability to evaluate similarities between key genetic drivers from a resected primary tumor and a PDA lesion that presented in the same patient 6 years later. Histological analysis and NGS can be used to understand potential differences and similarities between lesions and may be useful in future studies as predictive markers or to provide insight into resistance mechanisms (e.g., MYC amplification).

SSR-seq: Genotyping of microsatellites using next-generation sequencing reveals higher level of polymorphism as compared to traditional fragment size scoring.

Microsatellites (or simple sequence repeats, SSR) are widely used markers in population genetics. Traditionally, genotyping was and still is carried out through recording fragment length. Now, next‐generation sequencing (NGS) makes it easy to obtain also sequence information for the loci of interest. This avoids misinterpretations that otherwise could arise due to size homoplasy. Here, an NGS strategy is described that allows to genotype hundreds of individuals at many custom‐designed SSR loci simultaneously, combining multiplex PCR, barcoding, and Illumina sequencing. We created three different datasets for which alleles were coded according to (a) length of the repetitive region, (b) total fragment length, and (c) sequence identity, in order to evaluate the eventual benefits from having sequence data at hand, not only fragment length data. For each dataset, genetic diversity statistics, as well as F ST and R ST values, were calculated. The number of alleles per locus, as well as observed and expected heterozygosity, was highest in the sequence identity dataset, because of single‐nucleotide polymorphisms and insertions/deletions in the flanking regions of the SSR motif. Size homoplasy was found to be very common, amounting to 44.7%–63.5% (mean over all loci) in the three study species. Thus, the information obtained by next‐generation sequencing offers a better resolution than the traditional way of SSR genotyping and allows for more accurate evolutionary interpretations.