Vision Research 75 (2012) 117–129

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Vision Research

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Current mutation discovery approaches in Retinitis Pigmentosa ⇑ Ander Anasagasti a, Cristina Irigoyen c, Olatz Barandika a, Adolfo López de Munain a,b,d,e, Javier Ruiz-Ederra a, a Division of Neurosciences, Instituto Biodonostia, San Sebastián, Gipuzkoa, Spain b CIBERNED, Centro de Investigaciones Biomédicas en Red sobre Enfermedades Neurodegenerativas, Instituto Carlos III, Ministerio de Economía y Competitividad, Spain c Department of Ophthalmology, Hospital Universitario Donostia, San Sebastián, Spain d Department of Neurology, Hospital Universitario Donostia, San Sebastián, Spain e Department of Neurosciences, University of the Basque Country UPV-EHU, Spain article info abstract

Article history: With a worldwide prevalence of about 1 in 3500–5000 individuals, Retinitis Pigmentosa (RP) is the most Received 3 July 2012 common form of hereditary retinal degeneration. It is an extremely heterogeneous group of genetically Received in revised form 8 September 2012 determined retinal diseases leading to progressive loss of vision due to impairment of rod and cone pho- Available online 27 September 2012 toreceptors. RP can be inherited as an autosomal-recessive, autosomal-dominant, or X-linked trait. Non- Mendelian inheritance patterns such as digenic, maternal (mitochondrial) or compound heterozygosity Keywords: have also been reported. To date, more than 65 genes have been implicated in syndromic and non- Ciliopathy syndromic forms of RP, which account for only about 60% of all RP cases. Due to this high heterogeneity Next Generation Sequencing and diversity of inheritance patterns, the molecular diagnosis of syndromic and non-syndromic RP is very Photoreceptor Rod challenging, and the heritability of 40% of total RP cases worldwide remains unknown. However new Cone sequencing methodologies, boosted by the human genome project, have contributed to exponential plummeting in sequencing costs, thereby making it feasible to include molecular testing for RP patients in routine clinical practice within the coming years. Here, we summarize the most widely used state-of- the-art technologies currently applied for the molecular diagnosis of RP, and address their strengths and weaknesses for the molecular diagnosis of such a complex genetic disease. Ó 2012 Elsevier Ltd. All rights reserved.

1. Introduction hence the name to the disease. The onset, progression and severity of the disease is genetically determined in most cases and also The term Retinitis Pigmentosa (RP) encompasses a broad group influenced by the mode of inheritance. In extreme cases, patients of genetically determined retinal diseases caused by a large num- may present a rapid evolution over two decades, but in contrast, ber of mutations that result in rod photoreceptor cell death fol- other patients exhibit slow progression, which may never lead to lowed by gradual death of cone cells, eventually leading to blindness (Hamel, 2006). Nevertheless, symptoms typically start blindness. Thus, typical RP is also described as a rod-cone dystro- in the early teenage years and severe visual impairment occurs phy, in which loss of rod function exceeds the reduction in cone by ages 40–50 years (Sahni et al., 2011). sensitivity (Hamel, 2006; Hartong, Berson, & Dryja, 2006). RP is RP can be divided into two groups: non-syndromic RP in which the most common form of hereditary retinal degeneration with a RP is restricted to the eyes, without other systemic manifestations worldwide prevalence of about 1 in 3500–5000, with a total and syndromic RP in which patients present associated non-ocular of more than 1 million affected individuals (Chang et al., 2011; diseases, the latter representing 20–30% of total cases (Chang et al., Chizzolini et al., 2011; Collin et al., 2010). Affected individuals first 2011; Ferrari et al., 2011). The most common forms of syndromic RP experience defective dark adaptation (night blindness), followed are: Usher’s syndrome, which is characterized by RP and sensory- by reduction of the peripheral visual field (known as tunnel vision) neural hearing impairment, with or without vestibular dysfunction and sometimes, loss of central vision late in the course of the dis- (Williams, 2008) and the Bardet–Biedl syndrome, which is charac- ease. In the progression of RP symptoms, night blindness generally terized by RP with obesity, polydactyly, mental retardation, hypo- precedes tunnel vision by years or even decades. At the cellular gonadism and renal failure in some cases (Beales et al., 1999). The level, the retinal pigment epithelium is altered in most cases, Bardet–Biedl syndrome is due to mutations in at least 11 genes, presenting clusters of pigment within the retina of RP patients; with cases of triallelic and digenic inheritance (Hamel, 2006). On the basis of its inheritance pattern and prevalence, RP can be di- vided into three main groups: autosomal-dominant (30–40% of ⇑ Corresponding author. Address: Instituto Biodonostia, Paseo Dr. Begiristain s/n, E-20014 San Sebastián, Gipuzkoa, Spain. Fax: +34 943 00 62 50. cases), autosomal-recessive (50–60%) and X-linked (5–15%). Pa- E-mail address: [email protected] (J. Ruiz-Ederra). tients with no other affected relatives are typically autosomal

0042-6989/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.visres.2012.09.012 118 A. Anasagasti et al. / Vision Research 75 (2012) 117–129 recessive, although a few might represent new dominant muta- the current lack of large-scale genome sequencing studies aimed tions, instances of uniparental isodisomy or, for males, X-linked at disease gene discovery. However, this may soon change, consid- mutations, or even non-Mendelian inheritance patterns, such as ering that the cost of sequencing a genome at $1000 per individual digenic inheritance, compound heterozygosity or maternal (mito- is near to becoming a fact (Duggal, Ibay, & Klein, 2011). chondrial) inheritance (Audo, Bujakowska, et al., 2011; Audo, Since the discovery of the rhodopsin gene, the first one directly Lancelot, et al., 2011; den Hollander et al., 2007; Hartong, Berson, linked to RP (Dryja et al., 1990), almost 65 genes have been found & Dryja, 2006). to be associated with this disease. Considering recent advances in Most cases of RP are monogenic. More than 65 associated genes genomic sequencing, which are changing mutation discovery par- have been identified, 43 of which correspond to non-syndromic RP adigms, it seems reasonable to assume that the genetic cause of RP as of May 2012 (http://www.sph.uth.tmc.edu/retnet/). Most genes will be identified in 90–95% of cases in the near future. Identifica- for RP cause only a small proportion of cases, with the exception of tion and classification of all RP causing mutations will contribute to the rhodopsin (RHO), the USH2A and the RPGR genes which to- a better understanding of disease variants and will be central in or- gether cause 30% of all cases of RP (Daiger, Bowne, & Sullivan, der to provide improved diagnosis and prognosis for each patient. 2007; Hamel, 2006; Hartong, Berson, & Dryja, 2006; Musarella & This is particularly important when children, young adults or af- Macdonald, 2011; Pomares et al., 2010; Sergouniotis et al., 2011; fected women planning to have family are involved (Daiger, Waseem et al., 2007). However, these genes account for only about Bowne, & Sullivan, 2007; Ferrari et al., 2011; Hamel, 2006; Shintan- 60% of all RP patients, while heritability in about 40% of RP patients i, Shechtman, & Gurwood, 2009). remains unknown (Daiger, Bowne, & Sullivan, 2007; Ferrari et al., In this review, we summarize the most widely used, state- 2011; Sahni et al., 2011). For an update on genetic and genomic of-the-art technologies currently employed for the molecular information regarding complex genetic retinal disorders, several diagnosis of RP. We present their main features, advantages and databases are available, including: http://www.ensembl.org; disadvantages and assess their capabilities and limitations to http://www.ncbi.nlm.nih.gov; http://www.ncbi.nlm.nih.gov/gene; accomplish an accurate molecular diagnosis of such a complex http://www.ncbi.nlm.nih.gov/homologene; http://www.sph.uth.tmc. genetic disease. These techniques are presented in two main edu/retnet/; http://genome.ucsc.edu. groups: sequencing technologies and technologies specialized in Despite the fact that many technically diverse approaches are detecting genetic variants. being investigated for the treatment of RP, there is currently no standardized and efficient treatment for this disease. The discovery 2. Sequencing methods of the molecular basis of the disease has led to the development of several assays in animal models and more recently in human trials, 2.1. Chain-Termination or Sanger sequencing with promising results. More advanced lines of research in RP ther- apy include: the use of neurotrophic factors (Zhang et al., 2012); The Chain-Termination sequencing method or Sanger method gene therapy (Allocca et al., 2011; Jacobson et al., 2012; Pang (Sanger, Nicklen, & Coulson, 1977) has revolutionized molecular et al., 2011); retinal transplant (MacLaren et al., 2006; West biology, providing the backbone technology for DNA sequencing et al., 2012) and electronic prosthesis (Barry & Dagnelie, 2012; for almost three decades and has led to a number of monumental Zrenner et al., 2011). accomplishments, including the analysis of the whole human gen- Several factors have made the molecular characterization of RP ome sequence (Lander, 2011). This method is considered to be the a real challenge. These include the high number of genes and vari- first-generation technology, with latest technologies being denom- ants involved, as well as non-Mendelian inheritance patterns, such inated as Next-Generation Sequencing systems (NGSs) (Metzker, as incomplete penetrance, digenic or triallelic inheritance. To fur- 2010). ther complicate things, two different mutations in the same gene The Sanger method is performed in four separate reactions, in can generate different diseases and the same mutation in different each of which are included a DNA polymerase, specific DNA prim- individuals may cause distinctly different symptoms. For instance, ers flanking the target sequence and the four different types of although mutations in the rhodopsin gene are usually linked to deoxynucleotides (dNTPs). The key principle of this technique is dominant RP, a few rare rhodopsin mutations can cause recessive the use of chain-terminating dideoxynucleotide triphosphates RP. Finally, the fraction of disease-causing mutations varies with (ddNTPs). One of the four different types of radioactively labeled ethnicity and geography (Daiger, Bowne, & Sullivan, 2007; Ferrari ddNTPs is added to each reaction. Instead of having an –OH group, et al., 2011; Hamel, 2006; Stone, 2003). like dNTPs, ddNTPs have a hydrogen atom attached to the 30 Methods to determine DNA sequences were developed in the carbon, which causes the termination of the elongation reaction late 1970s by Frederick Sanger and Walter Gilbert, and have revo- due to their inability to form a phosphodiester bond with the next lutionized the science of molecular genetics. Development of the deoxynucleotide. Thus, DNA chain length in each reaction will Chain-Termination method in 1977, and especially publication of depend on how long the chain was when a ddNTP is randomly the first draft of the human genome in 2001 (Lander et al., 2001; incorporated. Once the four reactions have been completed, Venter et al., 2001), have boosted the blossoming of novel technol- high-resolution electrophoretic separation in a polyacrylamide ogies that deliver fast, inexpensive and accurate genome informa- gel is applied for separating these chains by length with a resolu- tion with unprecedented cost-effectiveness, leading to the tion of one nucleotide (Pettersson, Lundeberg, & Ahmadian, generation of what is known as Next Generation Sequencing 2009; Shendure & Ji, 2008). Finally, the gel is dried onto chroma- (NGS) technologies. Thanks to these new methods, the technical tography paper and exposed to X-ray film allowing direct reading challenges, time and cost involved in full or partial sequencing of of the DNA sequence, where a dark band in a lane indicates a the human genome have exponentially plummeted (Bowne, DNA fragment that is the result of chain termination after incorpo- Humphries, et al., 2011; Bowne, Sullivan, et al., 2011; Ferrari ration of a ddNTP (Fig. 1A). et al., 2011; Lander, 2011). For instance, the per-base cost of DNA In the early 1990s, a methodological improvement of the Sanger sequencing has dropped by 100,000-fold over the last decade and method, called Dye Termination sequencing, was introduced and the current generation of sequencing systems can read up to 250 has become the mainstay in automated sequencing. The main billion bases in a week compared with 25,000 in 1990 and 5 mil- improvement of this technique lies in the fact that each ddNTP is lion in 2000 (Lander, 2011; Service, 2006). High cost involved in labeled with a different fluorescent dye, allowing sequencing in a conventional methods of genome sequencing, are responsible of single reaction, rather than in four as in the Sanger method, with A. Anasagasti et al. / Vision Research 75 (2012) 117–129 119

Both strands of each size-selected, adapter-ligated DNA fragment are denatured and attached randomly, both forward and reverse, to nearby primers that are already covalently connected to a unique physical location in a solid surface called a flow cell (made up of a dense lawn of adaptor complementary sequences) (Pettersson, Lundeberg, & Ahmadian, 2009; Shendure & Ji, 2008). Each flow cell is divided into eight separate lanes which allow independent samples to be run simultaneously (Mardis, 2008). Thereafter, repeated cycles of bridge PCR amplification will generate directly on the surface, very high density colony-like local clusters (>10 million DNA clusters per each lane of the array), containing approximately 1000 copies of each single strand DNA fragment (Fig. 2)(Metzker, 2010; Pettersson, Lundeberg, & Ahmadian, 2009). Sequencing is then carried out using a proprietary cyclic revers- ible terminator-based method. This method enables detection of Fig. 1. Sanger and Dye Termination sequencing methods. Comparison between (A) single bases as they are incorporated into growing strands with a an autoradiograph obtained using the Sanger sequencing technique and (B) an modified DNA polymerase and a mixture of four nucleotides in electropherogram obtained by capillary electrophoresis using the Dye Termination each cycle (Shendure & Ji, 2008). Just like ddNTPs in Sanger sequencing technique. sequencing, a fluorescently labeled 30 reversible terminator base (RT-base) will stop amplification reactions when it is incorporated. a significant improvement in yield (Fig. 1B). Current sequencing At this time, after laser excitation, the emitted fluorescence (each platforms based on Dye Termination methodology are fully auto- RT-base emits a different fluorescence signal) from each cluster mated and use capillary electrophoresis in a massively parallel is captured by a sensitive CCD camera and the first base is identi- way, allowing the analysis of 384 DNA samples in a single run fied (Pareek, Smoczynski, & Tretyn, 2011). Non-incorporated nucle- which makes them faster, with implemented simplicity and cost- otides are washed away to allow incorporation of the next base. effectiveness and lower error rates, comparing to initial versions This is possible thanks to a cleavage enzyme that chops all the (Pettersson, Lundeberg, & Ahmadian, 2009; Shendure & Ji, 2008). blocking groups and extra molecules off (fluorophores and non- Sanger sequencing reads, including Dye Termination versions, incorporated bases), and turns the RT-base into a normally func- can reach up to 800–1000 base pairs (bp) with a per-base raw tioning nucleotide. Since all four reversible terminator-bound accuracy of near 100%. This accuracy is higher than that achieved dNTPs (A, C, T, G) are present as single, separate molecules during with NGS technologies, but has a much higher cost per kb, making each sequencing cycle, natural competition reduces the chance of this technology particularly inefficient for the generation of incorporation bias. The end result is true base-by-base sequencing. genome-size data sets (Table 1)(Fox, Filichkin, & Mockler, 2009; Illumina technology, currently the most cost-effective technol- Metzker, 2010; Shendure & Ji, 2008). ogy among all sequencing platforms, with the highest throughput, Currently, the main application of this technology is direct is able to generate 10-fold more sequence data for approximately sequencing or re-sequencing of amplified selected PCR products the same cost per run than other NGS technologies. The use of in- (Shendure & Ji, 2008), allowing analysis of genes and exons at dexes allows the multiplexing of 96 samples per lane, which is a the nucleotide level to elucidate disease-causing mutations. In fact, very useful feature when complex genetic diseases such as RP the Sanger technique is still the only method considered to be the are involved, and reduces significantly the cost per sample (Fox, gold standard for single read approaches in clinical sample testing Filichkin, & Mockler, 2009; Loman et al., 2012). Its main limitation and therefore is the method of choice for concluding molecular is its relatively short read length capacity of about 100 bases. This diagnosis (Garcia-Garcia et al., 2011; Janssen et al., 2011; McGee is probably due to dephasing and signal decay produced by incom- et al., 2010; Rajadhyaksha et al., 2010). plete incorporation of nucleotides, incomplete cleavage of fluores- cent labels or insufficient removal of reverse terminators. Longer reads are possible but at higher error rates. Thus, average raw error 2.2. Reverse dye-terminator (Illumina) sequencing rates are of the order of 0.75% per base for the Illumina GAIIx plat- form and 0.25% for the Illumina HiSeq 2000 platform, both of The Illumina sequencing system (formerly known as Solexa) which use a paired end sequencing approach (Minoche, Dohm, & combines the use of massively parallel clonal amplification with Himmelbauer, 2011; Quail et al., 2012). The dominant error type cyclic reversible dye-terminator sequencing (polymerase-based using Illumina technology is substitution, rather than insertions sequencing-by-synthesis). It is able to identify tens of billions of or deletions, but homopolymers are certainly less of an issue than bases per week in a single run, producing high quality sequence with other technologies (Elliott et al., 2012; Shendure & Ji, 2008). data with unprecedented levels of cost-effectiveness and through- Another alternative, such as ABI SOLiD sequencing technology, se- put (Pareek, Smoczynski, & Tretyn, 2011). This has facilitated the quences each nucleotide twice rather than as single bases because sequencing of complex genomes and comprehensive characteriza- it adds bases in pairs through a ligation-based sequencing tech- tion of the widest range of structural variants. It is currently one of nique. Thus, while Illumina will read ‘‘TAG’’ as ‘‘T’’, then ‘‘A’’, then the most widely adopted NGS platforms and recent applications of ‘‘G’’, ABI SOLiD reads it as ‘‘TA’’, ‘‘AG’’ (Koboldt et al., 2012). This this technology includes the diagnosis of RP (Audo et al., 2012; procedure produces a double sequencing of each base and conse- Bowne, Humphries, et al., 2011; Bowne, Sullivan, et al., 2011; quently significantly reduces reading error rates to about 0.15% Janssen et al., 2011; Tucker et al., 2011). per base. Using focused acoustic waves, the DNA sample is randomly Illumina technology can sequence 40,000,000 templates simul- sheared into fragments of 100–300 bp in length, and complemen- taneously with a raw accuracy of >98% and a throughput of 600 Gb tary sequences or adapters are ligated onto both ends of each frag- per run thanks to the paired-end reads approach, which consists of ment, increasing fragment size up to several hundred bp (Shendure a simple modification to the standard single-read DNA library & Ji, 2008). Adaptor-ligated DNA fragments are then size separated preparation. This facilitates reading both forward and reverse tem- by electrophoresis and a band between 200 and 300 bp is excised. plate strands of each cluster during one read, allowing alignment of 120 A. Anasagasti et al. / Vision Research 75 (2012) 117–129

Fig. 2. Schematic representation of the bridge PCR amplification process used in the Illumina sequencing platform. (A) DNA construct with adaptors in yellow and green, and sequence of interest in blue. (B) A dense lawn of adaptor complementary sequences (primers) covers the surface of a flow cell. (C) DNA constructs bind randomly to the primers attached to the flow cell. (D and E) DNA elongates along the bound DNA template. (F) The de novo synthesized sequence dissociates from the template strand. (G) The de novo synthesized sequence binds to an adjacent primer, generating a bridge, and the template strand is removed (arrow). (H) Bridge amplification takes place. (I) Both strands dissociate. (J) Non-attached extremes bind to new primers and a new bridge amplification cycle starts (modified from Illumina.com).

reads with unprecedented precision. The relative success of this each holding a different DNA fragment, are on top of a complemen- platform over other sequencing platforms, such as pyrosequencing tary metal–oxide semiconductor chip (CMOS), beneath which is an (Roche/454 GS FLX) or sequencing by oligonucleotide ligation and ion sensitive layer, a pH meter, below which is a proprietary Ion detection (ABI SOLiD), is also due to its lower consumable cost per Sensitive Field Effect Transistor Sensor (ISFET) which transmits Gb, which is approximately 1% of the cost of Sanger sequencing electrical current (Pareek, Smoczynski, & Tretyn, 2011; Rothberg (Pareek, Smoczynski, & Tretyn, 2011; Pettersson, Lundeberg, & et al., 2011). To sequence the template, the DNA to be deciphered Ahmadian, 2009). is first fragmented. Obtained fragments are then attached to adapt- ers, amplified by emulsion PCR and linked to a nano-well on the 2.3. Ion Semiconductor (Ion Torrent) sequencing chip (Rothberg et al., 2011). The device is flooded along with each of the four different unmodified bases (dNTPs), one type of base at Ion Torrent Semiconductor sequencing also known as pH-med- a time. When a dNTP complementary to the next nucleotide of the iated sequencing or silicon sequencing is a rapid (4.5 h per run for fragment is incorporated, pyrophosphate is released and a posi- 1 Gb output), simple, massively scalable and economical sequenc- tively charged hydrogen ion is generated. The charge from the ing system. Ion Torrent, rather than relying on optical or imaging ion changes the pH of the solution, which is detected by the ISFET technology, measures variations in pH induced by the release of ion sensor leading to a shift in voltage that is ultimately translated a positively charged hydrogen ion, coupled to nucleotide incorpo- into readout of the DNA being sequenced without scanning, cam- ration, a process coined as ‘‘sequencing by synthesis’’ (Glenn, eras or light mediation. If added dNTP in this cycle are not comple- 2011; Rothberg et al., 2011). Thus, semiconductor sequencing obvi- mentary, no incorporation and no biochemical reaction occurs. ates the need for optical methods, electromagnetic intermediates, Unattached dNTP molecules are washed out and another cycle be- specialized nucleotides or other technical challenges required in gins with a different type of base. When the ion sensor is excited, previous sequencing techniques (Glenn, 2011; Pareek, Smoczynski, electrical pulses are transmitted by the chip and the software dis- & Tretyn, 2011; Rothberg et al., 2011; Sanger, Nicklen, & Coulson, plays the produced data as an ionogram and the sequence can be 1977). read from it with no intermediate signal conversion (Fig. 3). The Ion Torrent device uses a high-density array of nano-scale The presence of a homopolymer stretch on the sequenced DNA wells to sequence in a massively parallel way. These nano-wells, causes incorporation of multiple dNTPs, with the corresponding A. Anasagasti et al. / Vision Research 75 (2012) 117–129 121

projects, including whole genome, exome or transcriptome sequencing (Life Technologies. Ion Proton sequencer and PGM sequencer Data Sheets, 2012). An advantage of ion semiconductor sequencing over many other approaches is that a simple run can be completed within hours, compared with previous NGS systems which have run times ranging from days up to 2 weeks. The fast performance associated with this technology has allowed the characterization of an Esche- richia coli strain in the early stages of a severe pathogenic outbreak by the rapid whole genome sequencing of this microorganism (Mellmann et al., 2011; Robison, 2011). Refining of this technology, mainly based on PES methodology, as described in Section 2.2, has allowed improved resolution of insertions and deletions, reducing reading errors by about 5-fold to 0.19% with a reported consensus accuracy of about 1 error per 1000 bases in 100 Mb. However, this accuracy and error data regarding the human genome are exclusively provided by the de-

Fig. 3. Ion Torrent. An ionogram corresponding to the first 55 flows from one well. vice manufacturers, with the exception of a recent study for the The Y-axis indicates the number of base pairs incorporated per well. The X-axis detection of disease-causing mutations in cystic fibrosis (Elliott indicates the flow number, with corresponding DNA base pairs. Note that colors are et al., 2012). Using semiconductor sequencing, they reported a assigned to bases in order to follow the Sanger sequencing nomenclature, since Ion false-positive call on the 2184delA in a CFTR locus in 35% of the Torrent technology is based on pH variation, with no fluorescence involved reads spanning this variant that lies in a homopolymer stretch of (unpublished results). seven adenines, which was misinterpreted as a point deletion. For this reason, the current high rate of false positives using semi- release of hydrogen ions in a single cycle. This causes a propor- conductor sequencing makes this technology more suitable for tional increase in pH and therefore in the electronic signal. How- other applications rather than for full gene sequencing, such as ever signals from those repeats with a 1 base difference are hotspot variant detection. Independent data regarding bacterial, difficult to discriminate. This limitation in accuracy of sequencing viral or non-human mammalian genome sequencing has been re- highly repetitive regions is shared by other techniques that detect ported by several groups (Howden et al., 2011; Mellmann et al., single nucleotide additions, such as pyrosequencing (Elliott et al., 2011; Miller et al., 2011; Vogel et al., 2012). 2012; Loman et al., 2012), and should be taken into consideration, Currently, several companies and universities are offering diag- since these regions can be important contributors to heritable dis- nostic services for RP, using NGS technology. Some examples in- eases (Gonzalez et al., 2007; Pareek, Smoczynski, & Tretyn, 2011). clude: The Casey Eye Institute Molecular Diagnostics Laboratory The initial scope of this technology was small scale, quick-turn- (Portland, Oregon. USA, https://www.ohsu.edu/xd/health/ser- around jobs like de novo bacterial artificial chromosomes (BACs), vices/casey-eye/diagnostic-services/cei-diagnostics/tests-we-offer. plasmids and microbial genome sequencing, microRNA sequencing cfm); the Center of Genomics and Transcriptomic CeGaT (Tuebin- or targeted re-sequencing, and was limited by the impossibility of gen, Germany, http://www.cegat.de/List-of-genes-(by-disease)_l= making long length reads (Glenn, 2011; Pareek, Smoczynski, & 1_41.html); the Centrum Medische Genetica (Ghent, Belgium, Tretyn, 2011). The recent launch of the Personal Genome Machine http://medgen.ugent.be/CMGG/onderzoek.php?topic_id=81); the (PGM) and Ion Proton sequencers with enhanced chip density (314, John and Marcia Carver Nonprofit Genetic Testing Laboratory (Iowa 316 or 318 chips) have improved read length from 100 bp to City, IOWA. USA, https://www.carverlab.org/faqs/disease-descrip- 200 bp, Mb per run yield (from 10 Mb to 1 Gb) and cost per Mb tions/rp); GeneDx (Gaithersburg, Maryland, USA, http://www. (about 5-fold) (Glenn, 2011; Robison, 2011). These two new Ion genedx.com/test-catalog/disorders/retinitis-pigmentosa-x-linked/); Torrent sequencers, although sharing semiconductor technology, Fundación Jimenez Diaz, http://www.fjd.es/instituto_investiga- are designed for different genetic approaches: the PGM sequencer cion/es/investigacion/genetica/genetica_genomica.html); and As- is recommended for small-genome sequencing, sequencing sets of per BioTech (http://www.asperbio.com/asper-ophthalmics). genes, ChIP-Seq, and gene expression approaches, whereas the Ion The main features of sequencing technologies reviewed here, as Proton sequencer is more suited to human genome sequencing well as other currently available NGS technologies, such as 454

Table 1 Key features of the principal Next Generation Sequencing technologies currently available.

Sanger 454 Pyroseq Illumina HiSeq 2000 ABI SOLiD 4 Ion Torrent Heliscope SMRT Sequencing Dideoxide Pyrosequencing Reversible Ligation-based Ion semiconductor Single-molecule Single-molecule method sequencing terminator sequencing sequencing sequencing sequencing chemistry Amplification Multiplex PCR Emulsión PCR Bridge PCR Emulsión PCR Emulsión PCR No amplification No amplification approach (single molecule) (single molecule) Gb per run (throughput) Single sequence/ 0.40–0.60 600a >100a sample 1a 21–35 100 Running time 2–3 h 7 h 8.5–11 daysa 7 daysa 4.5 ha 8 days 1 h Read lengths 1000 bp 400–500 bp 150 2bpa 35 75 bpa 200 bp 25–55 bp 3000–10,000 bp Costb 0.5c 84.00 0.035 0.04 0.93 0.45–0.60 Not determined

a Paired-end runs. b Estimated costs per Mb provided by manufacturers in USD. c Indicates cost per Kb. 122 A. Anasagasti et al. / Vision Research 75 (2012) 117–129 pyrosequencing, ABI SOLiD are summarized in Table 1. Heliscope for genome-wide association study (GWAS), which are efficient to and SMRT, also included in the Table 1, are briefly described in locate genes or loci that may harbor disease alleles (Duggal, Ibay, & Section 4. Klein, 2011; Hoffmann et al., 2012; Hunter et al., 2007; Levine et al., 2012; Rodenburg et al., 2012). Among the molecular diagnosis-oriented arrays, Arrayed Primer 3. Genetic variant detection methods Extension or APEX microarray technology, combines hybridization and four-color single-base primer extension reactions. This tech- 3.1. DNA microarray technology nology can genotype hundreds to thousands of genetic variations in parallel, including single base substitutions, deletions and inser- Microarray technology, also referred to as DNA chips, is a fast tions (Krjutskov et al., 2008; Pereiro et al., 2011; Pullat & Metspalu, and efficient high-throughput screening and validation technique 2008; Shumaker, Metspalu, & Caskey, 1996). successfully used in the diagnosis of several pathologies including The APEX reaction consists of a three-step reaction mechanism: glioblastoma, colorectal cancer, chronic lymphocytic leukemia, Idi- (1) amplification of targeted DNA which harbors mutations or SNPs opathic Generalized Epilepsy or autism (Dhawan et al., 2012; of interest; (2) hybridization between targeted DNA and primers Dougherty et al., 2012; Hochstenbach et al., 2011; Jasmine et al., attached to the beads (acting like probes) designed to hybridize 2012; Leone et al., 2012; Zhang et al., 2011). For a detailed review immediately adjacent to the loci of interest, stopping one base be- of its application in diagnosis see Keren and Le Caignec (2011) and fore the interrogated marker; and (3) single base enzymatic exten- Sato-Otsubo, Sanada, and Ogawa (2012). sion using a thermostable DNA polymerase together with four This technology, originally designed to detect genomic copy different terminator nucleotides each tagged with a different fluo- number variation (CNV) and to perform Single Nucleotide Poly- rophore. The newly incorporated nucleotide terminates the exten- morphism (SNP) genotyping, allows for whole genome analysis sion reaction and represents the base of interest. Then, four lasers of patient samples using a chip-based format. Current improve- (one at a time) are used to excite the different dyes and the light ments in DNA array features have contributed to making them very emitted is uses to interrogate the nature of the nucleotide in the popular for clinical research purposes. Analysis of thousands to position of interest (Pullat et al., 2008). It requires about 2.5– millions of SNPs in parallel can be performed, allowing large-scale 6 lg of genomic DNA and 2 days to perform the assay. genotyping studies with minimal hands-on processing per plate. APEX microarrays for the diagnosis of non-syndromic and syn- Applications for DNA arrays currently include clinical genetics dromic RP are commercially available. Non-syndromic RP variants and diagnosis, such as: gene expression profiling, alternative splic- which can be diagnosed include adRP (covering 414 variants in 16 ing detection, loss of heterozygosity testing, uniparental disomy or genes), arRP (594 variants in 19 genes) and X-linked RP (184 muta- DNA adenine methyltransferase identification (Bibikova et al., tions in 2 genes). Syndromic RP, such as Bardet–Biedl syndrome 2006; Gardina et al., 2006; Lin et al., 2004; Lindblad-Toh et al., (over 300 variants in 13 genes), or Usher syndrome (612 mutations 2000; Tucker et al., 2012; Vardhanabhuti et al., 2006). in 9 genes) can also be identified (Audo, Bujakowska, et al., 2011; Audo, Lancelot, et al., 2011; Audo et al., 2012; Avila-Fernandez 3.1.1. SNP or polymorphism microarrays et al., 2010; Dev Borman et al., 2012; Jaijo et al., 2010; Kimberling Variation in single nucleotide polymorphisms (SNPs) and copy et al., 2010; Mackay et al., 2011; Vozzi et al., 2011; Yzer et al., number variations (CNVs) within individuals play major roles in 2006). For an updated list of DNA arrays available see (http:// common human diseases. They may determine the difference www.asperbio.com/asper-ophthalmics). between affected and healthy individuals and are being There are currently two main manufacturers of GWAS-oriented extensively used as genetic markers (Almal & Padh, 2012; Vissers arrays. Both of these work on the same biochemical principal of & Stankiewicz, 2012). SNP or polymorphism microarrays are base-paring, but with some differences: Illumina arrays determine designed to detect most types of the SNP and CNV variants which SNP genotypes by the single-base extension technique, whereas have been previously ascertained through several major SNP Affymetrix arrays use single or multiple base mismatch hybridiza- discovery initiatives, such as NCBI dbSNP, the 1000 Genomes tion technology (LaFramboise, 2009; Perkel, 2008; Schaaf, Project, UCSC genome browser‘s database and the NHLBI Sequenc- Wiszniewska, & Beaudet, 2011). ing Project (http://www.ncbi.nlm.nih.gov/projects/SNP/; http:// In Affymetrix SNP arrays, probes of about 25 nucleotides long, www.1000genomes.org/; http://genome.ucsc.edu/ and http://www. representing all possible alleles at a polymorphic site (typically a nhlbi.nih.gov/resources/exome.htm; Hoffmann et al., 2012; Kim & pair), are attached to the 8 lm array beads. Once isolated, ampli- Misra, 2007; Rocha et al., 2006; Schaaf, Wiszniewska, & Beaudet, fied, fragmented and fluorescently labeled, the DNA target se- 2011; Smith, 2008; Sung et al., 2012). quence is applied to the chip, which will hybridize only to Microarrays are miniature devices made of silicon glass those probes which are perfectly complementary. The array is (1cm2) composed of millions of micrometer-scale beads or wells, scanned and the intensity of fluorescent signals is measured to each holding hundreds of thousands of identical copies of short quantify the relative amount of sample bound to each bead. The single-stranded DNA oligonucleotides – the probe – immobilized position of fluorescent signals within the array will identify which at a different and strategic position on the array. Probes exploit alleles are present in that particular genome sample. Affymetrix ar- the natural chemical attraction between DNA strands to hybridize rays are capable of determining in a single assay up to 1.8 million or to selectively anneal only to those DNA molecules with a perfect common and rare validated genetic variants, including insertion/ match. Therefore such arrays can be used to identify the presence deletions, which typically require 500 ng of genomic DNA and of a particular allele in the context of a single individual’s entire 3–4 days, for an average SNP reported call rate of >99% and an genome. average sample repeatability of >99.8% (Affymetrix Genome-Wide Attending to the purpose of the study, DNA arrays can be di- Human SNP Array 6.0. data sheet). This technology has been cho- vided in two main groups: those arrays used for the molecular sen for genotyping in projects related to retinal disorders (Bowne, diagnosis of specific genetic disorders, which typically require Humphries, et al., 2011; Bowne, Sullivan, et al., 2011; Gonzalez-del identification of hundreds to a few thousands of SNPs in parallel Pozo et al., 2011; Ostergaard et al., 2011; Roesch, Stadler, & Cepko, (Krjutskov et al., 2008; LaFramboise, 2009; Nevelin et al., 2012; 2012). Piñeiro-Gallego et al., 2011) and another group of arrays capable On the other hand, the Ilumina SNP array uses the single-base of performing parallel analysis of millions of SNPs, commonly used extension technique and a dual color channel approach to identify A. Anasagasti et al. / Vision Research 75 (2012) 117–129 123 and score up to 4.3 million markers per single DNA sample. Unlike Dufresne et al., 2006; Sergouniotis et al., 2011; Smith, Lu, & the Affymetrix assay, which utilizes one probe type per all possible Alvarado Bremer, 2010; Vossen et al., 2009). Transition of the alleles in a locus, the Illumina chip only uses a single probe – of double-stranded DNA molecule to its two single strands – DNA about 50 bases – per locus. In this case, the probe is designed to denaturation or melting – allows the study of DNA structure and hybridize immediately adjacent to the locus of interest, stopping composition by measuring the change of fluorescence intensity one base before the interrogated marker, thereby not containing per unit of time. HRM gives valuable information for mutation the SNP itself. By using this approach, there is no need to design screening, genotyping, methylation, microsatellite analysis and a different probe for each SNP in each locus, thereby increasing other research applications (Arthofer, Steiner, & Schlick-Steiner, the capacity of SNP genotyping (Schaaf, Wiszniewska, & Beaudet, 2011; Mader, Lukas, & Novak, 2008). 2011). HRM is faster (1.5–3 h), simpler and less expensive than other In Illumina SNP arrays, DNA target hybridizes to a locus-specific DNA variation analysis techniques, due to the lack of addition, pro- bead. Then, SNP locus-specific primers are extended in the pres- cessing or separation steps involved (Erali & Wittwer, 2010). Reac- ence of labeled dideoxynucleotides (biotin-labeled ddCTP and tion and analysis take place in the same instrument (closed-tube ddGTP, and dinitrophenyl group-labeled ddATP and ddUTP). These method) minimizing manipulation errors (Reed, Kent, & Wittwer, dideoxynucleotides are efficiently incorporated by polymerases 2007). These facts contribute to making HRM analysis more sensi- and detected in a dual-color assay. Bead fluorescence is analyzed tive and specific than other techniques, such as denaturing high using Illumina software for automated genotype calling, based on performance liquid chromatography (dHPLC), single-strand con- color and signal intensity (Steemers et al., 2006). With respect to formation polymorphism (SSCP) and restriction fragment length sample requirements, 750 ng of DNA is sufficient to process over polymorphism (RFLP) (Aguirre-Lamban et al., 2010; Joly et al., 500,000 SNP loci in 3 days, with an average SNP reported call rate 2011). Recent availability of advanced double-stranded DNA and reproducibility of about 100% (Illumina HumanOmni5-Quad (dsDNA)–binding dyes, along with next-generation real-time PCR BeadChip data Sheet). Recent studies were based on Illumina Bead- instrumentation and analysis software have increased its sensitiv- Chips to perform genome-wide homozygosity screening for arRP ity and accuracy and have improved the clinical potential of this and X-linked RP families (Kannabiran et al., 2012; Siemiatkowska technology. et al., 2011). HRM analysis starts with targeted PCR amplification in the pres- The main drawback of microarray-based genotyping, is that it is ence of a dsDNA-binding dye. Mutations can be detected at any only valid for known genetic variants, excluding newly discovered location in the amplicon, including those located right after the pri- SNPs. Both Illumina and Affymetrix are now producing arrays with mer sequences. As the PCR product gradually dissociates into sin- two components: one determined and another customizable, in gle strands, dye is released with a strong loss of fluorescence which researchers can incorporate a limited number of last minute emission related to the process (1000-fold decrease). Emitted discovered SNPs. This allows researchers to tailor the SNP chip to fluorescence is measured with specialized instrumentation which their needs, although at a significantly higher chip cost. generates a precise characteristic curve from a large number of Taqman OpenArrays (Applied Biosystems) offer a fully custom- fluorescent data points per change in temperature. By measuring izable array platform, which allows researchers to design custom the melting temperature (Tm: when 50% of the DNA is double- arrays to detect any new SNP. This technology uses the 50 exonu- stranded), amplicons that differ by only a single base compared clease activity of the Taq polymerase, together with two Taqman with reference wild type (WT) samples can be resolved (Dufresne probes to discriminate between the two alleles of an SNP. Taqman et al., 2006; Vossen et al., 2009). probes consist of two oligonucleotides, each complementary to one An interesting feature of HRM analysis is its power to detect of the two alleles of the SNP, with a fluorophore cova- all categories of variations, including substitutions, inversions, lently attached to the 50-end and a quencher at the 30-end. Quench- insertions and deletions, ranging from single to 63 bp mutations, er molecules suppress the fluorescence emitted by the fluorophore provided that they are small enough to be amplified by PCR, when excited by the cycler’s light, as long as the fluorophore and with forward and reverse primers bracketing the variation the quencher are close enough. During the annealing-extension (Bastien et al., 2008; De Leeneer et al., 2008; Dufresne et al., step, the probe hybridizes to the amplicon and polymerase breaks 2006; Kennerson et al., 2007; Reed, Kent, & Wittwer, 2007; van the union between the quencher and the DNA, resulting in an in- der Stoep et al., 2009). Thus, multi-exon deletions may remain crease of fluorescence due to physical separation of the quencher undetected by this technique (De Leeneer et al., 2008). from the fluorophore. The emitted fluorescence is directly propor- The sensitivity of heterozygote detection is remarkable, with tional to the amount of DNA and indicates the type of alleles that several studies reporting detection of all heterozygous mutations are present in the sample. The Taqman OpenArray platform con- examined (Audrezet et al., 2008; Kennerson et al., 2007; Lonie tains 3072 microscopic through-holes grouped in 48 subarrays of et al., 2006; Takano et al., 2008). Homozygous variants differ in 64 through-holes each, enabling the parallel genotyping of up to Tm from the WT samples whereas heterozygous variants produce 256 different SNPs per array. Running time for this assay is about a remarkable modification in curve shape, rather than in Tm. The 8 h from purified DNA to genotyping results and 125 ng of DNA latter are better resolved by using melting curve advanced plots, is required per sample (Hopp et al., 2010; Pomeroy et al., 2009; such as ‘‘Difference Plot’’, where small differences in melt curve Roberts et al., 2009). data are amplified (Liew et al., 2004). More recently, a fully customizable platform has become avail- Changes in Tm are base-specific. Class I (C/T; G/A) and II (C/A; able; the qBiomarker PCR array (SABioscience; Qiagen) allows the G/T) produce a greater change in Tm, with an average of >0.5 °C, complete design of an array with the SNPs chosen by the research- compared with class III (C/G), with an average of 0.2–0.5 °C or class er (patent pending). IV (A/T), with an average of <0.2 °C. Thus, mutations involving class III and IV are associated with a decreased sensitivity of homozy- 3.2. High Resolution Melting (HRM) analysis gote detection (Liew et al., 2004; Sergouniotis et al., 2011). One ap- proach that can be used to increase the power of resolution of High Resolution Melting (HRM) analysis is a fast and cost-effective single point mutations is to add WT DNA to control and unknown post-PCR analysis method that provides a rapid identification samples (at a 1:6 ratio) to create artificial heterozygotes, with all of genetic variation, based on biophysical measurement of the genotypes distinguished by quantitative heteroduplex analysis amplified DNA (Aguirre-Lamban et al., 2010; Carrillo et al., 2012; (Seipp, Herrmann, & Wittwer, 2010; Wittwer, 2009). Factors other 124 A. Anasagasti et al. / Vision Research 75 (2012) 117–129 than sequence that can generate small differences in Tm include: application of this genomic DNA technology includes SNP detec- genomic quality, primers design, amplicon length and PCR reagent tion, aneuploidy determination and DNA methylation analysis, as choice, including dye selection and ionic strength (Liew et al., well as RNA based applications such as RT-MLPA and mRNA profil- 2004). Amplicon lengths ranging between 100 and 300 bp, includ- ing (Diego-Alvarez et al., 2007; Slater et al., 2003). ing primers, are generally recommended for sequence variant MLPA is a multiplex PCR-based screening method designed to detection. determine in a single reaction tube the copy number of up to 50 Mutation discovery in human diseases has been the largest area DNA or RNA sequences, up to 96 samples in parallel in 24 h (Chung of HRM application, with investigations into autosomal dominant, et al., 2012; Stuppia et al., 2012; Vorstman et al., 2006). Unlike recessive and X-linked disorders (Cherbal et al., 2012; Kennerson standard multiplex PCR, where amplification using unique primer et al., 2007; Krypuy et al., 2007). To date, hundreds of mutations pairs for each fragment is required, MLPA reaction is performed in more than 60 genes have been analyzed with this technology, with only a single primer pair for all its probes, avoiding dimeriza- including genes associated with RP, such as C2ORF71, USH2A or tion or false priming issues. The principle of MLPA is based on the ABCA4 (Aguirre-Lamban et al., 2010; Sergouniotis et al., 2011; Xu identification of target sequences by hybridization of two oligonu- et al., 2011). We have recently found HRM analysis to be an appro- cleotides (fluorescently labeled MLPA probes) that can be joined to priate approach as a massive genetic screening method when com- each other by a ligation reaction, only when hybridized to the tar- bined with direct sequencing, being particularly suited for get sequence. Each oligonucleotide has a PCR primer attached to relatively small (<4 kb) RP genes with medium/high prevalence, facilitate posterior identification (Vorstman et al., 2006). Once such as USH3A, PRPF31 or ROM1 (Anasagasti et al., 2012)(Fig. 4). joined, MLPA sequences will then be exponentially amplified dur- ing PCR, providing a unique mixture of fragments ranging between 3.3. Multiplex Ligation Probe Amplification (MLPA) 65 and 500 bp in length, which can be identified and quantified by capillary electrophoresis and analyzed using an automatic sequen- The technique known as MLPA is an accurate, time-efficient and cer. This allows estimating the amount of product obtained by kit-based laboratory tool able to detect unusual copy number emission of a signal peak that can be visualized as an electrophero- changes in genomic sequences, with high sensitivity and specificity gram (Fernandez et al., 2005). Comparison of the peak probe pat- (Fernandez et al., 2005). This method is specialized in detecting tern emitted by the target sample with that emitted by a control large genomic rearrangements, i.e., deletions and duplications, sample enables the detection of abnormal probe signals that indi- including entire gene exons that frequently escape conventional cate variations in copy number, indicating either deletion or dupli- and novel laboratory detection methods, including NGS (Schouten cation of genomic regions of interest (Vorstman et al., 2006). A et al., 2002). Therefore, the MLPA technique has been frequently calculated ratio from this comparison that falls outside the range used in molecular diagnosis of patients with genetic disorders of 0.85–1.35 would indicate deletions or duplications of the sample caused by deletions or duplications in specific genes, such as Duch- sequence involved. enne Muscular Dystrophy (DMD), Charcot–Marie–Tooth disease or Zygosity can also be determined by ratio comparison, since both mental retardation (Dastur et al., 2011; Madrigal et al., 2007; homozygous deletions and duplications will produce greater ge- Marzese et al., 2008). Deletions and duplications represent about netic change and therefore greater change in ratios with respect 5% of all disease-causing mutations, and recent data show that this to the control sample than heterozygous deletions and duplica- might be of relevance in some cases of RP (Daiger, Bowne, & tions. Interpretation of data can be done using MLPA specific soft- Sullivan, 2007; Roux et al., 2011; Sullivan et al., 2006). Another ware (Vorstman et al., 2006). An important application of this

Fig. 4. High Resolution Melting (HRM) analysis. (A) HRM Difference Plot analysis, corresponding to exon 3 of the ROM1 gene. Two samples (in red, each in triplicate) differ clearly from control samples, suggesting the presence of a genetic variant. (B) Electropherogram obtained by direct sequencing corresponding to one of the hit samples shown in (A), confirming the presence of a heterozygote point deletion (arrow) within exon 3 of ROM1. Point deletion is identified by the irruption of a new fluorescence signal corresponding to the mutated strand that generates a shift in the reading frame (Anasagasti et al., 2012). A. Anasagasti et al. / Vision Research 75 (2012) 117–129 125

Lehmann-Horn et al., 2011), Marfan Syndrome (Faivre et al., 2010; Furtado et al., 2011), von Willebrand disease (Cabrera et al., 2011) or Aniridia (Lim et al., 2012) are commercially avail- able. As far as Retinitis Pigmentosa is concerned, there are several MLPA Kits marketed for genes associated with this disease, such as PRPF-31, PCDH15, RPGR, ABCA4, LCA or USH2A (Fig. 5)(Aller et al., 2010; Rose et al., 2011; Roux et al., 2011). Inconveniences of MLPA technology include the existence of only one supplier (MRC Hol- land), and the possibility of having an SNP under the probe. For a comparative summary of the main advantages and limita- tions of the different technologies described in the present review, see Table 2.

4. Future directions

Nowadays, we are experiencing a quantum shift in sequencing capacity, with benchtop devices being capable of unveiling genome sequences at unprecedented time and cost. It seems plausible thus to predict that molecular diagnosis for RP patients will become a routine procedure in clinical practice over the coming years. Nev- ertheless, due to the complexity of this eye disorder at many differ- ent levels, a number of important issues still need to be resolved. For instance, at least 40% of RP-related genes remain to be identi- fied, disease-causing mutations must be detected and classified, Fig. 5. Multiplex Ligation Probe Amplification (MLPA). Electropherogram corre- sponding to a region of the USH2A gene of an RP patient (top) and a non-affected mutation testing must become inexpensive, reliable and widely individual. Arrows show 3 regions with DNA insertions, corresponding to exons 71 available and, clinicians must become familiar with the molecular (left), 7 (middle) and 15 (right) (unpublished results). procedures used in order to communicate the relevant information to patients. In this respect, one of the limitations of NGS platforms is the interpretation of the huge amount of data generated. A mul- method is the determination of the relative ploidy of target sam- tidisciplinary network of clinicians, molecular biologists and bioin- ples. If an extra gene copy is present in the target sample, a 1.5-fold formaticians will have to be established, if we want to extract increase in emitted signal is predicted to occur, depending on the relevant biological information from the terabytes of data that zygosity of insertion. In contrast, if a gene copy in the target sample many laboratories are generating already. is missed, the emitted signal is expected to be 0.5 times the inten- New NGS platforms continue to appear on the market and will sity of the control probes in heterozygosis, with a complete lack of surely contribute to further changing the human genome sequenc- signal in homozygosis. ing paradigm. These include Helioscope, SMRT or Nanopore tech- Currently, MLPA kits containing all the necessary reagents to nologies. The principal advantages associated with these new perform diagnostic screening for many genetic disorders such as technologies, in addition to greater cost-effectiveness include: (1) Duchenne Muscular Dystrophy (DMD) (Dastur et al., 2011; Laing sequencing based on single molecule DNA, eliminating the need et al., 2011), Becker Muscular Dystrophy (BMD) (Lee et al., 2012; for PCR amplification, with a significant simplification of the

Table 2 Comparative summary of advantages and limitations of the main technologies for the detection of genetic variants and sequencing platforms.

Advantages Limitations Sanger Gold standard for single reads Poor quality in the first 15–40 bp, with deteriorating quality of sequencing traces after 700–900 bp Higher accuracy (99.999%) than NGS technologies Cost-ineffective, compared to NGS Long reads of1000 bp Illumina Highest throughput and most cost-effective technology among current Lower accuracy than Sanger and ABI SOLiD sequencing systems sequencing platforms Higher accuracy in homopolymer detection than other platforms Ion Torrent Easy to use, fast and low-cost instrument Low accuracy rates in homopolymer stretch >5 bp repeats No fluorescent procedures/detection systems needed Low discrimination power of similarly sized homopolymers Generates less noise data than other NGS platforms Microarray Detects all types of genetic variations, including large size deletions and Restricted to known mutations duplications Identification of millions of SNPs in parallel Customizable arrays are cost-ineffective Perform GWAS analysis with high accuracy HRM Highly cost-effective genetic screening technique Posterior direct sequencing is required in order to determine detected genetic variants All variation types detected, including small and mid-sized deletions and Work-intensive technique insertion MLPA Unique for large DNA rearrangements detection Dependent on availability of specific commercial kits Easy to perform and interpret Cost-ineffective Fast (results in <24 h) 126 A. Anasagasti et al. / Vision Research 75 (2012) 117–129 process and reduction of data generated; (2) read lengths are sig- Bowne, S. J., Humphries, M. M., Sullivan, L. S., Kenna, P. F., Tam, L. C., Kiang, A. S., nificantly higher than with Dye Termination sequencing, reaching et al. (2011). A dominant mutation in RPE65 identified by whole-exome sequencing causes retinitis pigmentosa with choroidal involvement. European up to 10,000 bp and (3) significant increase in data produced per Journal of Human Genetics, 19(10), 1074–1081. time invested. Note however that this information is based on data Bowne, S. J., Sullivan, L. S., Koboldt, D. C., Ding, L., Fulton, R., Abbott, R. M., et al. provided by the following manufacturers, with no independent (2011). 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