Next-generation sequencing technologies: An overview.

Since the days of Sanger sequencing, next-generation sequencing technologies have significantly evolved to provide increased data output, efficiencies, and applications. These next generations of technologies can be categorized based on read length. This review provides an overview of these technologies as two paradigms: short-read, or "second-generation," technologies, and long-read, or "third-generation," technologies. Herein, short-read sequencing approaches are represented by the most prevalent technologies, Illumina and Ion Torrent, and long-read sequencing approaches are represented by Pacific Biosciences and Oxford Nanopore technologies. All technologies are reviewed along with reported advantages and disadvantages. Until recently, short-read sequencing was thought to provide high accuracy limited by read-length, while long-read technologies afforded much longer read-lengths at the expense of accuracy. Emerging developments for third-generation technologies hold promise for the next wave of sequencing evolution, with the co-existence of longer read lengths and high accuracy.

Preliminary report of HLA (DNA) typing of Nigerians using sequence-specific primer technique.

AIMS AND OBJECTIVES: This communication is an attempt to present the experience and a preliminary report of results over a one-year period. PATIENTS AND METHODS: From December 2011 to December 2012, a prospective determination of the HLA types of 20 individuals referred to the Tissue Typing Laboratory of the Obafemi Awolowo University Teaching Hospitals Complex (OAUTHC), Ile-Ife was done. These consisted of prospective transplant recipients, their donors, and a migrant pair for kinship determination. DNA was extracted from the client's peripheral blood sample, using the QIAmp Blood DNA Mini kit, (Qiagen). PCR was done using OlerupR low-resolution PCR-SSP typing kit. The PCR product was resolved in 2% agarose gel, and the bands visualised under UV light. The HLA types were determined using provided tables and/or Helmberg software. Data were presented using descriptive statistics whileHLA antigen frequency (AF) was expressed in percentage and gene frequency (GF) was determined using square root method (1-(1-AF)1/2). RESULTS: A total of 20 individuals (13males and 7females) consisting of seven renal transplant recipients and seven prospective donors; a stem cell recipient and three donors and a migrant pair for kinship determination were typed. Age ranged from 4-65 years. 44 HLA alleles were detected, while HLA-A, B, C, DRB1 and DQB1 were 7, 10, 11, 8, 8 alleles respectively. The alleles were heterogeneous in distribution while 6 antigens (HLA-A*02, B*30, C*15, DRB1*03, DRB1*08 and DQB1*06) were having frequencies e"25%. CONCLUSION: This report confirms that DNA-based HLA typing is feasible locally, andit was observed that renal transplantation procedure is the most frequent indication. The HLA antigens observed to have very high frequencies (e"25% frequency) in this population were HLA-A*02, B*30, C*15, DRB1*03, DRB1*08 and DQB1*06. There is a strong need to develop a broad-based HLA data bank for Nigeria to further strengthening her transplantation programmes.

Hallazgo de patrones para péptidos-vacuna con capacidad de acople universal en moléculas de HLA-II / Identification of patterns for peptide-vaccines in universal capacity coupling HLA-II molecules / Identificagáo de padroes para peptídeos-vacina com capacidade de acoplamento universal em moléculas HLA-II

Partiendo del alineamiento múltiple de secuencias proteicas humanas obtenidas de las bases de datos del National Center of Biotechnology Information (NCBI) y su posterior análisis espacial tridimensional, se estableció la existencia de un patrón de acople universal para péptidos presentados por las moléculas de histocompatibilidad HLA-II (DR, DP y DQ), siendo una base para el diseño de vacunas proteicas. Estos patrones espaciales fueron claramente exhibidos por los residuos altamente conservados de los tres tipos de moléculas de HLA-II. La aplicación de este nuevo hallazgo permitió diseñar péptidos con mejores valores de acople péptido-HLA-II, que los generados por el péptido de acople universal conocido como CLIP (class Il-associated invariant chain peptide).

Starting from the multiple alignment of human protein sequences obtained from the NCBI database (National Center of Biotechnology Information) and subsequent three-dimensional spatial analysis, the existence of a pattern of universal coupling to peptides presented by MHC molecules HLA-II (DR, DP and DQ) was established, being a basis for the design of protein vaccines. These spatial patterns were clearly exhibited by highly conserved residues of the three kinds of HLA-II molecules. The application of this new finding made it possible to design peptides with better Peptide -HLA-II coupling values than those generated by the universal coupling peptide called CLIP (class II-associated invariant chain peptide).

FA partir do alinhamento múltiplo de sequéncias de proteínas humanas obtidas a partir das bases de dados do NCBI (National Center of Biotechnology Information) e análise espacial tridimensional subsequente, estabeleceu-se a existéncia de um padráo de acoplamento universal para peptídeos apresentados pelas moléculas de histocompatibilidade HLA-II (DR, DP e DQ), sendo uma base para o desenho de vacinas proteicas. Estes padroes espaciais foram claramente exibidos pelos residuos altamente conservados dos trés tipos de moléculas de HLA-II. A aplicagáo deste novo achado permitiu desenhar peptídeos com melhores valores de acoplamento peptídeo-HLA-II, do que aqueles gerados pelo peptídeo de acoplamento universal conhecido como CLIP (classe II-peptídeo associado a cadeia invariante).

Characterization of the novel HLA-C*07:195 allele in an Italian hematopoietic stem cell volunteer donor, detected by sequence-based typing.

The new allele shows one nucleotide change from C*07:02:01:01 at 351 nt from C to A, resulting in an amino acid change at codon 93 of exon 3 from H to Q.

Use of PCR with sequence-specific primers for high-resolution human leukocyte antigen typing of patients with narcolepsy.

BACKGROUND: Narcolepsy is a neurologic disorder characterized by excessive daytime sleepiness, symptoms of abnormal rapid eye movement (REM) sleep, and a strong association with HLA-DRB1*1501, -DQA1*0102, and -DQB1*0602. Here, we investigated the clinico-physical characteristics of Korean patients with narcolepsy, their HLA types, and the clinical utility of high-resolution PCR with sequence-specific primers (PCR-SSP) as a simple typing method for identifying DRB1*15/16, DQA1, and DQB1 alleles. METHODS: The study population consisted of 67 consecutively enrolled patients having unexplained daytime sleepiness and diagnosed narcolepsy based on clinical and neurological findings. Clinical data and the results of the multiple sleep latency test and polysomnography were reviewed, and HLA typing was performed using both high-resolution PCR-SSP and sequence-based typing (SBT). RESULTS: The 44 narcolepsy patients with cataplexy displayed significantly higher frequencies of DRB1*1501 (Pc= 0.003), DQA1*0102 (Pc=0.001), and DQB1*0602 (Pc=0.014) than the patients without cataplexy. Among patients carrying DRB1*1501-DQB1*0602 or DQA1*0102, the frequencies of a mean REM sleep latency of less than 20 min in nocturnal polysomnography and clinical findings, including sleep paralysis and hypnagogic hallucination were significantly higher. SBT and PCR-SSP showed 100% concordance for high-resolution typing of DRB1*15/16 alleles and DQA1 and DQB1 loci. CONCLUSIONS: The clinical characteristics and somnographic findings of narcolepsy patients were associated with specific HLA alleles, including DRB1*1501, DQA1*0102, and DQB1*0602. Application of high-resolution PCR-SSP, a reliable and simple method, for both allele- and locus-specific HLA typing of DRB1*15/16, DQA1, and DQB1 would be useful for characterizing clinical status among subjects with narcolepsy.

Use of PCR with Sequence-specific Primers for High-Resolution Human Leukocyte Antigen Typing of Patients with Narcolepsy

BACKGROUND: Narcolepsy is a neurologic disorder characterized by excessive daytime sleepiness, symptoms of abnormal rapid eye movement (REM) sleep, and a strong association with HLA-DRB1*1501, -DQA1*0102, and -DQB1*0602. Here, we investigated the clinico-physical characteristics of Korean patients with narcolepsy, their HLA types, and the clinical utility of high-resolution PCR with sequence-specific primers (PCR-SSP) as a simple typing method for identifying DRB1*15/16, DQA1, and DQB1 alleles. METHODS: The study population consisted of 67 consecutively enrolled patients having unexplained daytime sleepiness and diagnosed narcolepsy based on clinical and neurological findings. Clinical data and the results of the multiple sleep latency test and polysomnography were reviewed, and HLA typing was performed using both high-resolution PCR-SSP and sequence-based typing (SBT). RESULTS: The 44 narcolepsy patients with cataplexy displayed significantly higher frequencies of DRB1*1501 (Pc= 0.003), DQA1*0102 (Pc=0.001), and DQB1*0602 (Pc=0.014) than the patients without cataplexy. Among patients carrying DRB1*1501-DQB1*0602 or DQA1*0102, the frequencies of a mean REM sleep latency of less than 20 min in nocturnal polysomnography and clinical findings, including sleep paralysis and hypnagogic hallucination were significantly higher. SBT and PCR-SSP showed 100% concordance for high-resolution typing of DRB1*15/16 alleles and DQA1 and DQB1 loci. CONCLUSIONS: The clinical characteristics and somnographic findings of narcolepsy patients were associated with specific HLA alleles, including DRB1*1501, DQA1*0102, and DQB1*0602. Application of high-resolution PCR-SSP, a reliable and simple method, for both allele- and locus-specific HLA typing of DRB1*15/16, DQA1, and DQB1 would be useful for characterizing clinical status among subjects with narcolepsy.

Resolution of HLA class I sequence-based typing ambiguities by group-specific sequencing primers.

The increasing demand for allele-level human leukocyte antigen (HLA) typing has led the sequence-based typing (SBT) to become the preferred method. In turn, the steady increase in the number of HLA alleles driven by the adoption of SBT as the ultimate typing method leads to the ever increasing number of cis/trans ambiguities. Over the last few years, additional sequencing with the commercially available group-specific sequencing primers (GSSPs) has replaced sequence-specific primer-polymerase chain reaction and group-specific amplification as the means of resolving cis/trans ambiguities in many laboratories. Here we summarize our 3-year experience in designing and utilizing GSSPs for resolution of HLA class I ambiguities. The panel of GSSPs used in our laboratory includes 14 primers for HLA-A, 18 for HLA-B, and 13 primers for HLA-C. The panel resolves 99.9% of all ambiguities.

HLA-DR alleles associated with skin warts induced by human papillomavirus infection.

BACKGROUND: The skin wart is a benign proliferation of the skin and mucous, secondary to human papillomavirus (HPV) infection. OBJECTIVES: The objective of this study is to determine gene frequencies of HLA-DR alleles in Mexican patients with skin warts and compare them with those present in ethnically matched healthy subjects. METHODS: Fifty-two patients with clinically and histologically confirmed skin warts from the Dermatology Outpatient Clinic, with results of high-resolution DNA typing for HLA-DR polymorphism. RESULTS: HLA-DR3 and DR9 were increased (P = 0.0029, OR: 2.5, 95% CI: 1.3­4.7 and P = 0.0062, OR: 5.4, 95% CI: 1.4­19.5, respectively), and HLA-DR6 allele was found decreased (P = 0.0002). LIMITATIONS: The major histocompatibility complex contribution in the infection and elimination of the virus is not clear and perhaps also contributes to a series of events not well established yet. CONCLUSIONS: This study follows the preponderant role of class II genes in the susceptibility or resistance to the development of skin warts caused by HPV infection.

Improved KIR gene and HLA-C KIR ligand sequence-specific primer polymerase chain reaction genotyping using whole genome amplification.

Molecular analysis of genetic polymorphism for clinical or research purposes may be compromised by genomic DNA of limited quality and quantity. In this study, we have successfully tested the feasibility of using whole genome amplification (WGA) to allow genotyping for killer cell immunoglobulin-like receptor (KIR) genes and human leucocyte antigen (HLA)-C KIR ligand dimorphism on HLA-C. WGA was achieved by multiple displacement amplification (MDA) using bacteriophage phi29 polymerase. For KIR genotyping, a revised sequence-specific primer polymerase chain reaction protocol consisting of 23 primer pairs was used avoiding hitherto undetected cross-priming involving KIR2DL1, KIR2DS1, KIR3DL1 and KIR3DS1 alleles. Similarly, MDA-amplified genomic DNA was analyzed for the detection of the HLA-C KIR ligand groups C1 and C2, based on the amino acid K/N dimorphism in position 80.

Drug-induced liver injury: insights from genetic studies.

Drug-induced liver injury (DILI) is an increasing health problem and a challenge for physicians, regulatory bodies and the pharmaceutical industry, not only because of its potential severity and elusive pathogenesis but also because it is often inaccurately diagnosed, commonly missed entirely and more often not reported. The general view is that idiosyncratic DILI, which is not predictable whether based on the pharmacology of the drug or on the dose administered, is determined by the presence in the recipient of variants in, or expression of, genes coding for key metabolic pathways and/or the immune response, and the interaction of these genetic variants with environmental variables. Furthermore, idiosyncratic DILI is an example of a complex-trait disease with two or more susceptibility loci, as reflected by the frequency of genetic variants in the population often being higher than the occurrence of significant liver injury. Polymorphisms of bioactivation/toxification pathways via the CYP450 enzymes (Phase I), detoxification reactions (Phase II) and excretion/transport (Phase III), together with immunological factors that might determine DILI are reviewed. Challenges such as gene-trait association studies and whole-genome studies, and future approaches to the study of DILI are explored. Better knowledge of the candidate genes involved could provide further insight for the prospective identification of susceptible patients at risk of developing drug-induced hepatotoxicity, development of new diagnostic tools and new treatment strategies with safer drugs.

High incidence of type 1 diabetes mellitus during or shortly after treatment with pegylated interferon alpha for chronic hepatitis C virus infection.

BACKGROUND: Development of diabetes mellitus (DM) during or shortly after treatment with interferon alpha (IFN-alpha) in patients with chronic hepatitis C virus (HCV) infection has been reported sporadically. We prospectively screened for DM during and after IFN-alpha therapy for chronic HCV infection. METHODS: Blood glucose levels of patients with chronic HCV infection were routinely assessed at all outpatient visits during and after treatment with pegylated-IFN-alpha (Peg-IFN-alpha) and ribavirin (Riba). RESULTS: Between December 2002 and October 2005, 189 non-diabetic patients were treated with Peg-IFN-alpha/Riba, of whom five developed type 1 DM (2.6%), three type 2 DM (1.6%) and one an indeterminate type of DM. Classical symptoms of DM were present in three patients who developed DM shortly after cessation of Peg-IFN-alpha/Riba. In the other patients, symptoms of DM were either indistinguishable from side effects caused by Peg-IFN-alpha/Riba or absent. CONCLUSION: Our study showed a high incidence of type 1 DM during Peg-IFN-alpha/Riba therapy for chronic HCV infection. Symptoms of DM may be absent or mistaken for Peg-IFN-alpha/Riba-associated side effects. To diagnose DM without delay, we propose routine assessment of blood glucose at all outpatient visits during and after Peg-IFN-alpha/Riba treatment in chronic HCV patients.

External quality assessment of HLA-B*5701 reporting: an international multicentre survey.

OBJECTIVES: HLA-B*5701 strongly predicts abacavir hypersensitivity (HSR), but implementation of effective routine screening into clinical practice requires testing be practical and accurate. We tested the proficiency of HLA-B*5701 typing among laboratories using sequence-specific primer PCR. DESIGN AND METHODS: DNA panels (1 and 2) were distributed to seven laboratories (A to G) for blinded typing of the HLA-B*5701 allele. Panel 1 (n = 10 samples; n = 7 laboratories) included 3 positives and other closely related B17 subtypes (B*5702, B*5703, B*5704 and B*5801). Panel 2 (n = 96 samples; n = 4 laboratories) included 36 positives among a broad spectrum of other B alleles. Two laboratories (A and B) also submitted 96 routine samples, typed by the same methodology, to the reference centre for additional analysis by sequence-based typing. RESULTS: All laboratories correctly typed panel 1 for HLA-B*5701 carriage. Laboratories A, B and C identified HLA-B*5701 alleles in panel 2 with 100% sensitivity and 100% specificity. Laboratory D reported one false negative, reportedly due to a sampling error. The results obtained for routine samples typed by laboratories A and B and those generated by the reference laboratory using sequencing were fully concordant. CONCLUSIONS: Detection of HLA-B*5701 alleles among laboratories was 100% specific and 99.4% sensitive, indicating that participating HIV testing laboratories were currently offering effective primary screening to identify individuals at high risk of abacavir HSR. Accurate reporting of HLA-B*5701 status is critical for the safe administration of this drug and participation in quality assurance programmes by all sites who report HLA-B*5701 status should be promoted.

Genetic predisposition to rheumatoid arthritis in a Tuscan (Italy) ancient human remain.

Rheumatoid arthritis (RA) is currently believed to have originated in America, and after the discovery of this continent in 1492, to have been exported to the Old World. We evaluated the genetic predisposition to RA in the "Braids Lady" from Arezzo (Italy), a partially mummified woman's body dating back to the end of 1500 AD which presents the anatomical and pathological features of this disease. The study of the polymorphic HLA-DRB1 locus, which includes alleles strongly associated with RA onset, has received much attention over recent years, especially the loci codifying for the DR1 and DR4 antigens, widely represented in the Mediterranean population, and for DR14, widespread among Native Americans. Molecular analysis was performed on extracts of DNA from the mummy, firstly from histological bone sections and then from the whole bone. Two different HLA typing techniques, PCR-sequence-specific oligonucleotides (PCR-SSO) and PCR-sequence-specific primers (PCR-SSP), were employed to identify HLA-DRB alleles. Both genotyping methods showed that the "Braids Lady" carried the DRB1*0101 allele, the serological equivalent of the DR1 antigen. Although the possession of RA risk factor genes cannot be considered a diagnostic marker, the positive result of the Italian mummy for DRB1*0101 and the RA features present, support the idea that this pathology was present in the Old World from at least the mid-16th century. A pathogenetic hypothesis of RA which might well explain its worldwide diffusion is the "molecular mimicry", resulting from a cross-reactive antibody response between certain microbial antigens and shared epitopes of specific HLA-DR1, DR4 and DR14 susceptibility alleles, the frequency of which varies among different ethnic groups.

A one-step real-time PCR assay for detection of DQA1*05, DQB1*02 and DQB1*0302 to aid diagnosis of celiac disease.

Celiac disease is an autoimmune disorder that develops after dietary exposure of the small intestine to gluten peptides in cereals. Celiac disease has a strong genetic component associated with HLA-DQ2 and HLA-DQ8, and testing for absence of these genetic markers is useful when serological tests and biopsies are indeterminate, as it renders celiac disease highly unlikely. We have developed a new real-time PCR assay, using sequence-specific primers (PCR-SSP) and TaqMan probes, for detection of DQB1*05, DQB1*02 (coding for DQ2) and DQB1*0302 (coding for DQ8). PCR amplification and detection of DQ2 and DQ8 was accurately and unambiguously performed from genomic DNA isolated from cell lines and human DNA. Amplification was scored digitally, without laboratory manipulation of amplified PCR products and with a higher accuracy than PCR-SSP. This assay should increase accuracy and throughput, and reduce risks of contamination in laboratories where testing for HLA DQ2 and DQ8 is performed as part of diagnosis of celiac disease.

Validation of sensitive human leukocyte antigen-sequence-specific primer and probe typing in forensic DNA examination.

Validation studies were carried out with the commercially available HLA typing kit using a PCR-SPP (sequence-specific primer and probe) technique. This technique has made it possible to type class I (HLA-A and -B) and class II (HLA-DRB1 and -DQB1) alleles at low-resolution level with total 10 ng of template DNA, in addition to amplify directly from various forms of blood samples without DNA isolation procedure. Experimental examinations with bloodstains smeared on cotton cloth that were a week to 3 months old, bloodstains on gauze stored for 18 years, and buccal cells revealed that this HLA-SPP typing kit is a sensitive and reliable method for forensic investigations.

A high-resolution polymerase chain reaction-sequence-specific primer HLA-B*27 typing set and its application in routine HLA-B27 testing.

We designed a set of 35 polymerase chain reaction sequence-specific primers (PCR-SSP) in 29 SSP mixtures to assign 29 HLA-B*27 4-digit level alleles (B*2701-B*2721 and B*2723-B*2730). This was used in conjunction with our 41 PCR-SSP primer mixture low-resolution HLA-B typing set to fully differentiate B*27 from all other HLA-B alleles. Successful typing set validation used 521 B*27 samples covering 13 (B*2701-B*2710 and B*2712, B*2717, B*2723) alleles. The distribution of B*27 alleles was determined in a random population of 4020 local blood donors and the use of PCR-SSP B*27 typing in our routine flow cytometry-based HLA-B27/B2708 typing strategy is described.

Real-time PCR using fluorescent resonance emission transfer probes for HLA-B typing.

HLA genotyping by polymerase chain reaction (PCR) has some inherent labor-intensive and effort-demanding limitations. To overcome them, we have developed a real-time PCR with hybridization probes approach able to obtain a medium-low resolution HLA-B genotyping with fewer tubes and probes and with a shorter time requirement. Our strategy used 18 simultaneous reactions amplifying HLA-B alleles and an internal control. Monitorization of both amplifications in each tube is performed by the simultaneous application of two fluorescent resonance emission transfer probes: the first probe, different for each tube, is specific for the HLA-B locus and the second probe detects the control gene. A medium-low resolution (300 HLA-B allelic groups) typing is obtained for each sample by analyzing the melting curve patterns. Because some alleles may be determined without using the complete set of reactions, we present an alternative strategy: a first round of seven reactions and, according to the result, a second (or third) round of PCRs to solve the ambiguities. This method was validated in pretyped clinical samples and the results were completely concordant. Moreover, fewer ambiguous results were obtained. In summary, we present a new, faster, and more accurate method than currently used PCR techniques to type HLA-B alleles.

Probe selection algorithm for oligonucleotide array-based medium-resolution genotyping.

Medium-resolution genotyping has the goal of distinguishing different subgroups instead of each element in a group. An oligonucleotide array provides an inexpensive, high-throughput method to identify differences in DNA sequence among individuals, which is fundamental for genotyping. As the cost and difficulty of designing and fabricating the oligonucleotide array dramatically increase with the number of probes used, it is therefore important to have a design with a minimum number of probes meeting the requirement of medium-resolution genotyping. The first algorithm for designing and selecting probes for oligonucleotide array-based medium-resolution typing is reported. The goal in deriving the algorithm was to select a minimum number of probes from a large probe set on the premise of minimum loss of resolution. The algorithm, which was based on entropy, conditional entropy and mutual information theory, was used to select the minimum number of probes from a large probe set. The algorithm was tested on a human leukocyte antigen (HLA) sequence data set Thirty probes were selected from 390 probes for HLA-A, and 60 probes were selected from 767 probes for HLA-B. Although the number of probes was reduced by almost ten times, the distinguishability was reduced only a little, by 0.45% (from 99.90% to 99.45%) for HLA-A and 0.27% (from 99.84% to 99.57%) for HLA-B, respectively. This is a satisfactory and practical result.

Identification of new HLA-C alleles by polymerase chain reaction-sequence specific oligonucleotide typing: Cw*0314 and Cw*1511*.

In this article, we report two new human leukocyte antigen-C (HLA-C) alleles, HLA-Cw*0314 and Cw*1511, which were identified during routine tissue typing of donors for the Australian Bone Marrow Donor Registry and Australian Cord Blood Bank. HLA-Cw*0314 shows six codon changes in exon 3 compared to Cw*030401 and shares some sequence homology with Cw*07 alleles. Cw*1511 has two nucleotide changes compared with Cw*150201 in exon 2, both resulting in amino acid changes in the protein sequence.