Comparative tolerance of two massively parallel sequencing systems to common PCR inhibitors.

Human remains can be severely affected by the environment, and the DNA may be damaged, degraded, and/or inhibited. In this study, a DNA sample (at 1 ng DNA target input in triplicate) was spiked with five concentrations of five inhibitors (humic acid, melanin, hematin, collagen, and calcium) and sequenced with both the HID-Ion AmpliSeq™ Library Kit and ID panel on the Ion PGM™ System and the ForenSeq DNA Signature Prep Kit on the MiSeq FGx™. The objective of this study was to compare the baseline tolerance of the two sequencing chemistries and platforms to common inhibitors encountered in human remains recovered from missing person cases. The two chemistries generally were comparable but not always susceptible to the same inhibitors or at the same capacity. The HID-Ion AmpliSeq™ Library Kit and ID panel and the ForenSeq DNA Signature Prep Kit both were susceptible to humic acid, melanin, and collagen; however, the ForenSeq kit showed greater inhibition to melanin and collagen than the AmpliSeq™ kit. In contrast, the ForenSeq kit was resistant to the effects of hematin and calcium, whereas the AmpliSeq™ kit was highly inhibited by hematin. Short tandem repeats (STRs) and single nucleotide polymorphisms (SNPs) showed the same trend among inhibitors when using the ForenSeq kit. Generally, locus read depth, heterozygote allele balance, and the numbers of alleles typed were inversely correlated with increasing inhibitor concentration. The larger STR loci were affected more so by the presence of inhibitors compared to smaller STR amplicons and SNP loci. Additionally, it does not appear that sequence noise is affected by the inhibitors. The noise percentage, however, does increase as the inhibitor concentration increases, due to the decrease in locus read depth and not likely because of chemistry effects.

Novel missense mutation in the bZIP transcription factor, MAF, associated with congenital cataract, developmental delay, seizures and hearing loss (Aymé-Gripp syndrome).

BACKGROUND: Cataract is a major cause of severe visual impairment in childhood. The purpose of this study was to determine the genetic cause of syndromic congenital cataract in an Australian mother and son. METHOD: Fifty-one genes associated with congenital cataract were sequenced in the proband using a custom Ampliseq library on the Ion Torrent Personal Genome Machine (PGM). Reads were aligned against the human genome (hg19) and variants were annotated. Variants were prioritised for validation by Sanger sequencing if they were novel, rare or previously reported to be associated with paediatric cataract and were predicted to be protein changing. Variants were assessed for segregation with the phenotype in the affected mother. RESULT: A novel likely pathogenic variant was identified in the transactivation domain of the MAF gene (c.176C > G, p.(Pro59Arg)) in the proband and his affected mother., but was absent in 326 unrelated controls and absent from public variant databases. CONCLUSION: The MAF variant is the likely cause of the congenital cataract, Asperger syndrome, seizures, hearing loss and facial characteristics in the proband, providinga diagnosis of Aymé-Gripp syndrome for the family.

Alternative genome sequencing approaches of SARS-CoV-2 using Ion AmpliSeq Technology.

A thorough understanding of SARS-CoV-2 genetic features is compulsory to track the ongoing pandemic across multiple geographical locations of the world. Thermo Fisher Scientific USA has developed the Ion AmpliSeq SARS-CoV-2 Research Panel for the targeted sequencing of SARS-CoV-2 complete genome with high coverage and lower error rate. In this study an alternative approach of complete genome sequencing has been validated using different commercial sequencing kits to sequence the SARS-CoV-2. Amplification of cDNA with the SARS-CoV-2 primer pool was performed separately using two different master mixes: 2X environmental master mix (EM) and Platinum™ PCR SuperMix High Fidelity master mix (PM) instead of 5X Ion AmpliSeq™ HiFi Mix whereas NEBNext® Fast DNA Library Prep Set for Ion Torrent™ kit was used as an alternative to Ion AmpliSeq Library Kit Plus for other reagents. This study demonstrated a successful procedure to sequence the SARS-CoV-2 whole genome with average ∼2351 depth and 98.1% of total the reads aligned against the reference sequence (SARS-CoV-2, isolate Wuhan-Hu-1, complete genome). Although genome coverage varied, complete genomes were retrieved for both reagent sets with a reduced cost. This study proposed an alternative approach of high throughput sequencing using Ion torrent technology for the sequencing of SARS-CoV-2 in developing countries where sequencing facilities are low. This blended sequencing technique also offers a low cost protocol in developing countries like Bangladesh.

Toxoplasma gondii infection and peripheral-blood gene expression profiling of older people reveals dysregulation of cytokines and identifies hub genes as potential therapeutic targets.

Infections of humans with the protozoan parasite Toxoplasma gondii (T. gondii) can lead to the disease's development, even in an asymptomatic status. However, the mechanisms that result in these clinical outcomes after infection are poorly understood. This study aimed to explore the molecular pathogenesis of toxoplasmosis-related inflammation through next-generation sequencing, to assess RNA expression profiles in peripheral blood from 5 female patients with chronic toxoplasmosis and 5 healthy female controls. All plasma samples were analyzed for anti-Toxoplasma IgG and IgM antibody titers by using electrochemiluminescence. Detection of acute and chronic toxoplasmosis was carried out using the ELISA IgG avidity. We evaluated the levels of INF-γ, IL-2, IL-12, TNF-α, IL-10, and IL-1ß in culture supernatants of Peripheral Blood Mononuclear Cells infected with Toxoplasma lysate antigen (TLA) prepared with tachyzoites of strain T. gondii RH. Differential expression analysis was performed using DESeq2, pathway and enrichment analysis of DEGs was done on WEB-based Gene SeT AnaLysis Toolkit (WebGestalt) and Protein-protein interaction was carried out using NetworkAnalyst with STRING. In older people with chronic asymptomatic infection, a significant difference in the levels of inflammatory cytokines INF-γ and IL-2 was observed compared to seronegative individuals. Our results revealed differences in the regulation of critical biological processes involved in host responses to chronic T. gondii infection. Gene ontology analysis revealed several biologically relevant inflammatory and immune-related pathways.

Inflammatory gene expression profiling in peripheral blood from patients with Alzheimer's disease reveals key pathways and hub genes with potential diagnostic utility: a preliminary study.

BACKGROUND: Alzheimer's disease (AD) is an age-related neurodegenerative disease caused by central nervous system disorders. Late-onset Alzheimer disease (LOAD) is the most common neurodegenerative disorder worldwide. Differences at the expression level of certain genes, resulting from either genetic variations or environmental interactions, might be one of the mechanisms underlying differential risks for developing AD. Peripheral blood genome transcriptional profiling may provide a powerful and minimally invasive tool for the identification of novel targets beyond Aß and tau for AD research. METHODS: This preliminary study explores molecular pathogenesis of LOAD-related inflammation through next generation sequencing, to assess RNA expression profiles in peripheral blood from five patients with LOAD and 10 healthy controls. RESULTS: The analysis of RNA expression profiles revealed 94 genes up-regulated and 147 down-regulated. Gene function analysis, including Gene Ontology (GO) and KOBAS-Kyoto Encyclopedia of DEGs and Genomes (KEGG) pathways indicated upregulation of interferon family (INF) signaling, while the down-regulated genes were mainly associated with the cell cycle process. KEGG metabolic pathways mapping showed gene expression alterations in the signaling pathways of JAK/STAT, chemokines, MAP kinases and Alzheimer disease. The results of this preliminary study provided not only a comprehensive picture of gene expression, but also the key processes associated with pathology for the regulation of neuroinflammation, to improve the current mechanisms to treat LOAD.

Forensic characterization of Brazilian regional populations through massive parallel sequencing of 124 SNPs included in HID ion Ampliseq Identity Panel.

Use of Massive Parallel Sequencing (MPS) techniques has been investigated by forensic community aiming introduction of such methods in routine forensic casework analyses. Interesting features presented by MPS include high-throughput, ability to simultaneous genotyping of significant number of samples and forensic markers, workflow automation, among others. Emergence of single nucleotide polymorphism (SNP) as forensic relevant markers was facilitated in this process, since concurrent typing of larger marker sets is necessary for obtaining same levels of individual discrimination provided by other marker categories. In this context, HID Ion Ampliseq Identity Panel is a commercial solution with forensic purposes comprising simultaneous analysis of 90 highly informative autosomal SNPs and 34 Y -chromosome superior clade SNPs for male lineage haplotyping. SNP typing can be obtained with smaller amplicons, and this panel was designed for efficient processing of critical or challenging forensic samples. In this work, a sample of 432 individuals from all five Brazilian geopolitical regions was evaluated with this panel, in order to access feasibility of this panel use in a national basis. Results obtained for all five regions, including forensic parameters, show that this marker set can be efficiently employed for Brazilian nationals in human identification or kinship determination applications, due to high levels of genetic discriminative information content displayed by Brazilians. Interpopulation comparison studies were executed among Brazilian regional populations and 26 worldwide populations, in order to access genetic stratification occurrence. Some levels of population structure were identified, and impact on database design was discussed. Y-chromosome haplotyping of Brazilian samples revealed high levels of European ancestry in Brazilian male lineages, and utility of haplotyping in real forensic casework is addressed. Finally, genotyping and sequencing efficiency with this panel were addressed, as an effort to appraise the adequacy of this panel use in Brazilian national forensic demands.

Validation and Clinical Applications of a Comprehensive Next Generation Sequencing System for Molecular Characterization of Solid Cancer Tissues.

Identification of somatic molecular alterations in primary and metastatic solid tumor specimens can provide critical information regarding tumor biology and its heterogeneity, and enables the detection of molecular markers for clinical personalized treatment assignment. However, the optimal methods and target genes for clinical use are still being in development. Toward this end, we validated a targeted amplification-based NGS panel (Oncomine comprehensive assay v1) on a personal genome machine sequencer for molecular profiling of solid tumors. This panel covers 143 genes, and requires low amounts of DNA (20 ng) and RNA (10 ng). We used 27 FFPE tissue specimens, 10 cell lines, and 24 commercial reference materials to evaluate the performance characteristics of this assay. We also evaluated the performance of the assay on 26 OCT-embedded fresh frozen specimens (OEFF). The assay was found to be highly specific (>99%) and sensitive (>99%), with low false-positive and false-negative rates for single-nucleotide variants, indels, copy number alterations, and gene fusions. Our results indicate that this is a reliable method to determine molecular alterations in both fixed and fresh frozen solid tumor samples, including core needle biopsies.

Brazilian forensic casework analysis through MPS applications: Statistical weight-of-evidence and biological nature of criminal samples as an influence factor in quality metrics.

Real forensic casework biological evidence can be found in a myriad of different conditions and presenting very distinct features, including key elements such as degradation levels, the nature of biological evidence, mixture presence, and surface or substrate deposition, among others. Technical protocols employed by forensic DNA analysts must consider such characteristics in order to improve the chances of successfully genotyping these materials. MPS has been used as a very useful tool for forensic sample processing and genetic profile generation. However, it is not completely clear how different features encountered with real forensic samples impact sequencing quality and, consequently, profile accuracy and reliability. In this context, the present study analyzes a set of 47 real forensic casework samples, obtained from semen, saliva, blood and epithelial evidence, as well as reference oral swabs, aiming to evaluate the impact of a sample's biological nature in profiling success. All DNA extracts from samples were standardized according to sample conditions, as assessed by traditional forensic profiling methods (real-time PCR quantitation and capillary electrophoresis-coupled STR fragment analysis). Samples were separated into groups according to their biological nature, and the resultant sequencing quality was evaluated through a series of well-established statistical tests, applied specifically to six different MPS quality metrics. The results showed that certain groups of samples, especially epithelial and (to a lesser extent) saliva samples, exhibited significantly lower quality in terms of some of the evaluated metrics. A number of reasons for such unexpected behavior are discussed. In addition, a series of calculations was performed to assess the weight of genetic evidence in Brazilian samples, and reflexes in data analysis and national allele frequency database construction are discussed. Overall, the results indicate that a unified national allele frequency database can be used nationwide. Besides this, MPS genetic profiles obtained from samples with particular biological origins may benefit from meticulous manual review, and visual inspection could be important as an additional step to avoid genotyping errors or misinterpretation, leading to more trustworthy and reliable results in real criminal forensic casework analysis.

Molecular drug resistance profiles of Mycobacterium tuberculosis from sputum specimens using ion semiconductor sequencing.

The increasing burden of multidrug resistant (MDR)-TB, defined by resistance to rifampin (RFP) and isoniazid (INH), and extensively drug resistant-TB, defined by MDR-TB with additional resistance to fluoroquinolones (FQs) and more than one second-line injectable drug, is a serious impediment to global TB control. We evaluated the feasibility of full-length gene analysis including inhA, katG, rpoB, pncA, rpsL embB, eis, and gyrA using a semiconductor NGS with the Ion AmpliSeq TB panel to directly analyse 34 sputum specimens confirmed by phenotypic DST: INH, RFP, ethambutol (EMB), pyrazinamide (PZA), amikacin, kanamycin, streptomycin (SM), FQs including ofloxacin, moxifloxacin, and levofloxacin. The molecular drug resistance profiles showed "very good" and "substantial" strength of agreement for the phenotypic DST results of RFP and EMB, PZA, SM, FQs resistance with specificities of 96%, and 88%, 97%, 100% and sensitivities of 100%, and 88%, 60%, 67%, respectively. The strength of agreement for the detection of resistance to INH was "substantial", compared between katG mutation and phenotypic INH only. Ion semiconductor NGS could make possible detection of several uncommon or novel amino acid changes in the full coding regions of these eight genes. However, molecular drug resistant profile should be complemented and validated by subsequent phenotypic DST studies at the same time.

Determining Genotypic Drug Resistance by Ion Semiconductor Sequencing With the Ion AmpliSeq™ TB Panel in Multidrug-Resistant Mycobacterium tuberculosis Isolates.

BACKGROUND: We examined the feasibility of a full-length gene analysis for the drug resistance-related genes inhA, katG, rpoB, pncA, rpsL, embB, eis, and gyrA using ion semiconductor next-generation sequencing (NGS) and compared the results with those obtained from conventional phenotypic drug susceptibility testing (DST) in multidrug-resistant Mycobacterium tuberculosis (MDR-TB) isolates. METHODS: We extracted genomic DNA from 30 pure MDR-TB isolates with antibiotic susceptibility profiles confirmed by phenotypic DST for isoniazid (INH), rifampin (RIF), ethambutol (EMB), pyrazinamide (PZA), amikacin (AMK), kanamycin (KM), streptomycin (SM), and fluoroquinolones (FQs) including ofloxacin, moxifloxacin, and levofloxacin. Enriched ion spheres were loaded onto Ion PI Chip v3, with 30 samples on a chip per sequencing run, and Ion Torrent sequencing was conducted using the Ion AmpliSeq TB panel (Life Technologies, USA). RESULTS: The genotypic DST results revealed good agreement with the phenotypic DST results for EMB (Kappa 0.8), PZA (0.734), SM (0.769), and FQ (0.783). Agreements for INH, RIF, and AMK+KM were not estimated because all isolates were phenotypically resistant to INH and RIF, and all isolates were phenotypically and genotypically susceptible to AMK+KM. Moreover, 17 novel variants were identified: six (p.Gly169Ser, p.Ala256Thr, p.Ser383Pro, p.Gln439Arg, p.Tyr597Cys, p.Thr625Ala) in katG, one (p.Tyr113Phe) in inhA, five (p.Val170Phe, p.Thr400Ala, p.Met434Val, p.Glu812Gly, p.Phe971Leu) in rpoB, two (p.Tyr319Asp and p.His1002Arg) in embB, and three (p.Cys14Gly, p.Asp63Ala, p.Gly162Ser) in pncA. CONCLUSIONS: Ion semiconductor NGS could detect reported and novel amino acid changes in full coding regions of eight drug resistance-related genes. However, genotypic DST should be complemented and validated by phenotypic DSTs.

Simple and efficient germline copy number variant visualization method for the Ion AmpliSeq™ custom panel.

BACKGROUND: Recent advances in molecular genetic analysis using next-generation sequencing (NGS) have drastically accelerated the identification of disease-causing gene mutations. Most next-generation sequencing analyses of inherited diseases have mainly focused on single-nucleotide variants and short indels, although, recently, structure variations including copy number variations have come to be considered an important cause of many different diseases. However, only a limited number of tools are available for multiplex PCR-based target genome enrichment. METHODS: In this paper, we reported a simple and efficient copy number variation visualization method for Ion AmpliSeq™ target resequencing data. Unlike the hybridization capture-based target genome enrichment system, Ion AmpliSeq™ reads are multiplex PCR products, and each read generated by the same amplicon is quite uniform in length and position. Based on this feature, the depth of coverage information for each amplicon included in the barcode/amplicon coverage matrix file was used for copy number detection analysis. We also performed copy number analysis to investigate the utility of this method through the use of positive controls and a large Japanese hearing loss cohort. RESULTS: Using this method, we successfully confirmed previously reported copy number loss cases involving the STRC gene and copy number gain in trisomy 21 cases. We also performed copy number analysis of a large Japanese hearing loss cohort (2,475 patients) and identified many gene copy number variants. The most prevalent copy number variation was STRC gene copy number loss, with 129 patients carrying this copy number variation. CONCLUSION: Our copy number visualization method for Ion AmpliSeq™ data can be utilized in efficient copy number analysis for the comparison of a large number of samples. This method is simple and requires only easy calculations using standard spread sheet software.

Syndromic hearing loss molecular diagnosis: Application of massive parallel sequencing.

Syndromic hearing loss accounts for approximately 30% of all cases of hearing loss due to genetic causes. Mutation screening in known genes is important because it potentially sheds light on the genetic etiology of hearing loss and helps in genetic counseling of families. In this study, we describe a customized Ion AmpliSeq Panel, specifically designed for the investigation of syndromic hearing loss. The Ion AmpliSeq Panel was customized to cover the coding sequences of 52 genes. Twenty-four patients were recruited: 17 patients with a clinical diagnosis of a known syndrome, and seven whose clinical signs did not allow identification of a syndrome. Of 24 patients sequenced, potentially causative mutations were found in nine, all of which belonged to the group with a previous clinical diagnostic and none in the group not clinically diagnosed. We were able to provide conclusive molecular diagnosis to six patients, constituting a diagnostic rate of 25% (6/24). In the group of patients with a suspected clinical diagnosis, the diagnostic rate was 35% (6/17). Of the nine different mutations identified, three are novel, and were found in patients with Waardenburg, Treacher Collins and CHARGE syndromes. Since all patients with a conclusive molecular diagnosis through this panel had a previous suspected clinical diagnosis, our results suggest that this panel was more effective in diagnosing this group of patients. Therefore, the panel demonstrated effectiveness in molecular diagnosis when compared to others in the literature, especially for patients with a defined clinical diagnosis.

Investigating kinship of Neolithic post-LBK human remains from Krusza Zamkowa, Poland using ancient DNA.

We applied an interdisciplinary approach to investigate kinship patterns and funerary practices during the middle Neolithic. Genetic studies, radiocarbon dating, and taphonomic analyses were used to examine two grave clusters from Krusza Zamkowa, Poland. To reconstruct kinship and determine biological sex, we extracted DNA from bones and teeth, analyzed mitochondrial genomes and nuclear SNPs using the HID-Ion AmpliSeq™ Identity panel generated on Illumina and Ion Torrent platforms, respectively. We further dated the material (AMS 14C) and to exclude aquatic radiocarbon reservoir effects, measures of carbon and nitrogen stable isotopes for diet reconstruction were used. We found distinct mitochondrial genomes belonging to haplogroups U5b2a1a, K1c and H3d in the first grave cluster, and excluded maternal kin patterns among the three analyzed individuals. In the second grave cluster one individual belonged to K1a4. However, we could not affiliate the second individual to a certain haplogroup due to the fragmented state of the mitochondrial genome. Although the individuals from the second grave cluster differ at position 6643, we believe that more data is needed to fully resolve this issue. We retrieved between 26 and 77 autosomal SNPs from three of the individuals. Based on kinship estimations, taking into account the allelic dropout distribution, we could not exclude first degree kin relation between the two individuals from the second grave cluster. We could, however, exclude a first degree kinship between these two individuals and an individual from the first grave cluster. Presumably, not only biological kinship, but also social relations played an important role in the funerary practice during this time period. We further conclude that the HID-Ion AmpliSeq™ Identity Panel may prove useful for first degree kin relation studies for samples with good DNA preservation, and that mitochondrial genome capture enrichment is a powerful tool for excluding direct maternal relationship in ancient individuals.

Molecular Characterization of Somatic Alterations in Dukes' B and C Colorectal Cancers by Targeted Sequencing.

Despite global progress in research, improved screening and refined treatment strategies, colorectal cancer (CRC) remains as the third most common malignancy. As each type of cancer is different and exhibits unique alteration patterns, identifying and characterizing gene alterations in CRC that may serve as biomarkers might help to improve diagnosis, prognosis and predict potential response to therapy. With the emergence of next generation sequencing technologies (NGS), it is now possible to extensively and rapidly identify the gene profile of individual tumors. In this study, we aimed to identify actionable somatic alterations in Dukes' B and C in CRC via NGS. Targeted sequencing of 409 cancer-related genes using the Ion AmpliseqTM Comprehensive Cancer Panel was performed on genomic DNA obtained from paired fresh frozen tissues, cancer and normal, of Dukes' B (n = 10) and Dukes' C (n = 9) CRC. The sequencing results were analyzed using Torrent Suite, annotated using ANNOVAR and validated using Sanger sequencing. A total of 141 somatic non-synonymous sequence variations were identified in 86 genes. Among these, 64 variants (45%) were predicted to be deleterious, 38 variants (27%) possibly deleterious while the other 39 variants (28%) have low or neutral protein impact. Seventeen genes have alterations with frequencies of ≥10% in the patient cohort and with 14 overlapped genes in both Dukes' B and C. The adenomatous polyposis coli gene (APC) was the most frequently altered gene in both groups (n = 6 in Dukes' B and C). In addition, TP53 was more frequently altered in Dukes' C (n = 7) compared to Dukes' B (n = 4). Ten variants in APC, namely p.R283∗, p.N778fs, p.R805∗, p.Y935fs, p.E941fs, p.E1057∗, p.I1401fs, p.Q1378∗, p.E1379∗, and p.A1485fs were predicted to be driver variants. APC remains as the most frequently altered gene in the intermediate stages of CRC. Wnt signaling pathway is the major affected pathway followed by P53, RAS, TGF-ß, and PI3K signaling. We reported the alteration profiles in each of the patient which has the potential to affect the clinical decision. We believe that this study will add further to the understanding of CRC molecular landscape.

Revealing the challenges of low template DNA analysis with the prototype Ion AmpliSeq™ Identity panel v2.3 on the PGM™ Sequencer.

Forensic scientists frequently have to deal with the analysis of challenging sources of DNA such as degraded and low template DNA (LtDNA). The capacity to genotype difficult biological traces has been facilitated by emerging technologies. Massive parallel sequencing (MPS) on microchip among other technologies promises high sensitivity and discrimination power. In this study we evaluated the combined use of the Quantifiler(®) Trio DNA Quantification Kit with the prototype Ion AmpliSeq™ Identity panel v2.3 and PGM™ platform in LtDNA samples. Coverage, allele balance, allele drop-out/in, consistency and variance were assessed. Overall, the results showed a great level of performance and consistency in terms of genotyping capability even under the most challenging conditions, making it possible to obtain consistent SNP profiles with 31 pg of DNA and partial informative profiles with as little as 5 pg or with severely degraded DNA. In addition, we demonstrated that the stochastic effects observed in some samples are due to the amplification of the library rather than sequencing. Based on our data, we proposed general recommendations for the analysis of casework samples starting from the use of quantification data, which proved to be critical in deciding whether to process the samples via STR (short tandem repeat) analysis or SNP MPS. In our experience, the use of the prototype Ion AmpliSeq™ Identity panel v2.3 has revealed a new applicable solution for processing LtDNAs. This approach provides users with an additional tool for analysis of traces that either would not give informative results with conventional STR-based techniques.

Next generation sequencing of SNPs using the HID-Ion AmpliSeq™ Identity Panel on the Ion Torrent PGM™ platform.

The HID-Ion AmpliSeq™ Identity Panel (the HID Identity Panel) is designed to detect 124-plex single nucleotide polymorphisms (SNPs) with next generation sequencing (NGS) technology on the Ion Torrent PGM™ platform, including 90 individual identification SNPs (IISNPs) on autosomal chromosomes and 34 lineage informative SNPs (LISNPs) on Y chromosome. In this study, we evaluated performance for the HID Identity Panel to provide a reference for NGS-SNP application, focusing on locus strand balance, locus coverage balance, heterozygote balance, and background signals. Besides, several experiments were carried out to find out improvements and limitations of this panel, including studies of species specificity, repeatability and concordance, sensitivity, mixtures, case-type samples and degraded samples, population genetics and pedigrees following the Scientific Working Group on DNA Analysis Methods (SWGDAM) guidelines. In addition, Southern and Northern Chinese Han were investigated to assess applicability of this panel. Results showed this panel led to cross-reactivity with primates to some extent but rarely with non-primate animals. Repeatable and concordant genotypes could be obtained in triplicate with one exception at rs7520386. Full profiles could be obtained from 100pg input DNA, but the optimal input DNA would be 1ng-200pg with 21 initial PCR cycles. A sample with ≥20% minor contributor could be considered as a mixture by the number of homozygotes, and full profiles belonging to minor contributors could be detected between 9:1 and 1:9 mixtures with known reference profiles. Also, this assay could be used for case-type samples and degraded samples. For autosomal SNPs (A-SNPs), FST across all 90loci was not significantly different between Southern and Northern Chinese Han or between male and female samples. All A-SNP loci were independent in Chinese Han population. Except for 18loci with He <0.4, most of the A-SNPs in the HID Identity Panel presented high polymorphisms. Forensic parameters were calculated as >99.999% for combined discrimination power (CDP), 0.999999724 for combined power of exclusion (CPE), 1.390×1011 for combined likelihood ratio (CLR) of trios, and 2.361×106 for CLR of motherless duos. For Y-SNPs, a total of 8 haplotypes were observed with the value of 0.684 for haplotype diversity. As a whole, the HID Identity Panel is a well-performed, robust, reliable and high informative NGS-SNP assay and it can fully meet requirements for individual identification and paternity testing in forensic science.

Determining Genotypic Drug Resistance by Ion Semiconductor Sequencing With the Ion AmpliSeq™ TB Panel in Multidrug-Resistant Mycobacterium tuberculosis Isolates

BACKGROUND: We examined the feasibility of a full-length gene analysis for the drug resistance-related genes inhA, katG, rpoB, pncA, rpsL, embB, eis, and gyrA using ion semiconductor next-generation sequencing (NGS) and compared the results with those obtained from conventional phenotypic drug susceptibility testing (DST) in multidrug-resistant Mycobacterium tuberculosis (MDR-TB) isolates. METHODS: We extracted genomic DNA from 30 pure MDR-TB isolates with antibiotic susceptibility profiles confirmed by phenotypic DST for isoniazid (INH), rifampin (RIF), ethambutol (EMB), pyrazinamide (PZA), amikacin (AMK), kanamycin (KM), streptomycin (SM), and fluoroquinolones (FQs) including ofloxacin, moxifloxacin, and levofloxacin. Enriched ion spheres were loaded onto Ion PI Chip v3, with 30 samples on a chip per sequencing run, and Ion Torrent sequencing was conducted using the Ion AmpliSeq TB panel (Life Technologies, USA). RESULTS: The genotypic DST results revealed good agreement with the phenotypic DST results for EMB (Kappa 0.8), PZA (0.734), SM (0.769), and FQ (0.783). Agreements for INH, RIF, and AMK+KM were not estimated because all isolates were phenotypically resistant to INH and RIF, and all isolates were phenotypically and genotypically susceptible to AMK+KM. Moreover, 17 novel variants were identified: six (p.Gly169Ser, p.Ala256Thr, p.Ser383Pro, p.Gln439Arg, p.Tyr597Cys, p.Thr625Ala) in katG, one (p.Tyr113Phe) in inhA, five (p.Val170Phe, p.Thr400Ala, p.Met434Val, p.Glu812Gly, p.Phe971Leu) in rpoB, two (p.Tyr319Asp and p.His1002Arg) in embB, and three (p.Cys14Gly, p.Asp63Ala, p.Gly162Ser) in pncA. CONCLUSIONS: Ion semiconductor NGS could detect reported and novel amino acid changes in full coding regions of eight drug resistance-related genes. However, genotypic DST should be complemented and validated by phenotypic DSTs.