http://blog.genohub.com/11-top-next-generation-sequencing-blogs/
https://en.m.wikipedia.org/wiki/Cancer_genome_sequencing
NGS FOR FRIENDS WITH A FUTURE P,J,C AND PP, for all colorectal, xxx neg breast, melanoma, and cup cancer of unknown primary
FOR CLAIRE XXX NEGATIVE
with a mutation will not have a significant family history to warrant testing.95,96Therefore, these individuals would only be tested if other more local guidelines are used such as young age of onset or triple negative breast tumour pathology. These groups are likely to benefit from a more readily available NGS approach. There is a similar situation in high-grade serous ovarian cancer in that around 50% of women with serous ovarian cancer who h
Still alive and feeling wonderful, full of hope love and gratitude after the most dangerous, daring immuno tace ever attemoted, ever achieved, of course this is a fantasy. It would never be permitted in a world of deadly red tape, of authorities that read blogs, and harrass and gaol the worlds most amazing doctors. so for the record, for the authorities everything in this blog is fiction. after all, rats cannot type, can they? how could this be real? do cancer miracles happen? when the stage 4 s all die painful meta static deaths in a world where 10 million euro european funded laboratory makes dendtric cells fueled with hope. how do you reconcile all the suffering with all the hope and not go crazy?
I dreamed I won another nobel prize, I have lost count of how many I have now, and I still don't have a phd, I have something much much better. LIFE! and an illness that evolves as I do and in the process allows amazing clinical fantasies to occur, far beyond the scientists wildest dreams and the regulators worst nightmares. you see meta static cancer has viable treatment options NOW, remission as close to the nearest STAR. have you touched a star recently? I do every breathe, every second of my miracle, that's where the unlimited energy I need to heal comes from. That's another way of saying god.
I ask you? am I the luckiest?
So I sip peppermint tea at the hotel of the lion in duderstadt, this morning I gave 200ml blood for dc vaccine 17, I got good to the point hallway time with nesslehut senior, and prof osmers while the blonde sexy vampire sucked me dry while the genius that's prof osmers listened, learned, digested and spat out the clinical pearls, just like nessle senior did in the hallway.
the pearls fuel my hope. the details SECRET ALAS!
who needs hour long padded out consulting room discussion, I give a clinical synopsis in a minute, we discuss, brain storm, I get the clinical pearls. the doctors race to rooms full of patients, they have a very very busy day ahead saving lives, dangling the carrot, MIRACLE REMISSION.
this rat is locked on the never ending, circular wheel, the treadmill of cancer survival.
like the donkey climbing everest, with a big juicy orange carrot constantly hanging in front of him, driving him onwards, uppwards to the summit, to his dreams, to his god.
likewise I had a carrot juice and tomatoe to help refill the blood stolen by the vampire today.
these are the worlds best immunotherapists without doubt. they will get the nobelest prize. is not one miracle , one life saved, one life extended, worth all the nobel prizes ever awarded.
I really believe I am the luckiest man alive.
I have a new 3cm liver met that sparkles like the sun in the recent pet scan, in the upper left lobe near the heart. its gods greatest gift, sounds strange, read on, better read this link and think hard
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3219767/
so what's ngs? are your cancer mutations on the cell surface? are all ngs equal, and for that matter what micro rna are you using to enhance your immune response to get your tcells focused on survival like I am. so maybe this new met is a gift, now I have lived long enough, to unravel the genetic message that's the secret code for long tern durable remission.
these ngs allows, remission strategies, combo antibody strategies, we now have the tools, this rats very very happy, and gets a lot of joy being so far ahead of the pack. I maybe the fastest rat ever to have lived.
day to day the survival strategy evolves.
the worlds best oncologist said, I have something truly special for you, see me 4pm tomorrow, that was yesterday, read on about the best ngs solution and the stage 4 melanoma miracle
so yesterday I did a super ipt indulin potentiated therapy as a followup to tace 32, whose focus was to embolise and shutdown liver met.
re cancer markers, my bet pseudo progression as 199 has not moved, I have the highest lymphocyte count ever, the high cea tumour destruction .
I looked at the pet scan, besides the bright sun in my liver, what did I see?
something rare, wonderful, indeed god divided, billions of dendritic cells in my left upper arm swarming around the dc vaccine site, engaging in a billion simultaneous facebook conversations, messaging each other. the messages are the dcs sharing the antigens that continue to save me.
this pet scan, 3 days post last dc vaccine was a stroke of genius, now we can see immune shadows moving around our bodies like the hand of god. maybe ill call that 3d image the shadowy sun, as I have these hope filled shadows all around the vaccine site, and a vivid sun in the liver that again is brightest on the edges where the glucose avid immune cells destroy the destroyer.
its time for another sauna and swim.
remember the 6 peritoneal mets, have all been cleared by immuno tace, dc vaccine, pdt, and intraperitoneal oncolytic viral therapy enhanced by gcmaf. fixing peritoneal disease, the hardest, most challenging by far, but easy when you understand its an immunological organ and that conventional medicine is ignorant in I extreme, alas life saving peritonecomies will be viewed as barbaric, especially now subcutenous removab exists, I is another option.
I have to discuss organising a clinical trial with vogl, this is the future for many cancer patients, its my reality, my invention, my achievement, its in my present and its your present, if you have the courage, money and skill to make it happen.
the it, is survive.
i begged the pet scan prof to see me, to discuss imaging
i need to see my german pet scan guru asap.
i need to find a german university who wants to study the worlds first human pseudo lymph node, to think within me now, in my liver is a therapy technique that could save millions of lives and the immune cells are sipping peopermint tea with me.
its party, festival time in duderstadt this weekend, the kids running races keep me sane, keep me anchored to reality, here comes more runners. i blow my little family a kiss, i miss them, but i live , i learn. i blow the big family a kiss also.
it is what it is, AMEN!
this life is BLISS!
ps in asco, they told the story of nivolumab patients who had progression who were sent home to die, they followed up a year later, again the corpses answered the calls. these corpses were miracles. immunological memory the key.
FOR PIP FROM THINKING HARD, GOOD FOR CUP CANCERS
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Journal ListClin Biochem Revv.32(4); 2011 NovPMC3219767
Clin Biochem Rev. 2011 Nov; 32(4): 177–195.
PMCID: PMC3219767
Next-Generation Sequencing for Cancer Diagnostics: a Practical Perspective
Cliff Meldrum,1,4,† Maria A Doyle,2,† and Richard W Tothill3,*
Author information ► Copyright and License information ►
This article has been cited by other articles in PMC.
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Abstract
Next-generation sequencing (NGS) is arguably one of the most significant technological advances in the biological sciences of the last 30 years. The second generation sequencing platforms have advanced rapidly to the point that several genomes can now be sequenced simultaneously in a single instrument run in under two weeks. Targeted DNA enrichment methods allow even higher genome throughput at a reduced cost per sample. Medical research has embraced the technology and the cancer field is at the forefront of these efforts given the genetic aspects of the disease. World-wide efforts to catalogue mutations in multiple cancer types are underway and this is likely to lead to new discoveries that will be translated to new diagnostic, prognostic and therapeutic targets. NGS is now maturing to the point where it is being considered by many laboratories for routine diagnostic use. The sensitivity, speed and reduced cost per sample make it a highly attractive platform compared to other sequencing modalities. Moreover, as we identify more genetic determinants of cancer there is a greater need to adopt multi-gene assays that can quickly and reliably sequence complete genes from individual patient samples. Whilst widespread and routine use of whole genome sequencing is likely to be a few years away, there are immediate opportunities to implement NGS for clinical use. Here we review the technology, methods and applications that can be immediately considered and some of the challenges that lie ahead.
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Overview
Over the past six years we have witnessed a revolution in sequencing technologies that has already had a profound impact on our understanding of genetics and genome biology. In a research setting, NGS has been widely implemented for de novo genome sequencing, DNA resequencing, transcriptome sequencing and epigenomics. These research efforts have forged the way in the development of new protocols (both molecular and bioinformatic contexts) and have been instrumental in gaining an understanding of the major strengths and weaknesses of this technology. From a clinical perspective there is great potential for NGS in the management and treatment of human health. Immediate and significant impact will come from either replacement or augmentation of existing technologies for genetic screening purposes. Some striking examples of its clinical use include prenatal testing for the detection of chromosomal aneuploidy in foetal DNA,1 identification of rare genetic variants associated with monogenic Mendelian disorders2–4 and efficient detection of either inherited or somatic mutations in cancer genes.5,6
As cancer is a genetic disease driven by heritable or somatic mutations, new DNA sequencing technologies will have a significant impact on the detection, management and treatment of disease. Next-generation sequencing is enabling worldwide collaborative efforts, such as the International Genome Consortium (ICGC)7and The Cancer Genome Atlas (TCGA) project,8 to catalogue the genomic landscape of thousands of cancer genomes across many disease types. Several early reports from individual studies contributing to these consortia have already been published.9–11 These discoveries will ultimately lead to a better understanding of disease pathogenesis, bridging to a new era of molecular pathology and personalised medicine.12 It is easy to imagine that soon every patient will have both their constitutional and cancer genomes sequenced, the latter perhaps multiple times in order to monitor disease progression, thus enabling an accurate molecular subtyping of disease and the rational use of molecularly guided therapies. Many molecular pathology laboratories are now considering the sequencing platforms, methods and additional equipment required for making the transition to NGS. Here we review the current sequencing technology, applications and bioinformatics with special consideration given to the development of clinical DNA sequencing.
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Next-Generation Sequencing Technology
NGS broadly describes those technologies that share the ability to massively parallel sequence millions of DNA templates. The terms second-generation and third-generation sequencing are also used synonymously to describe the evolution of sequencing technology from the first-generation dideoxy ‘Sanger’ sequencing. To achieve massive parallel sequencing, second-generation platforms employ the clonal amplification of DNA templates on a solid support matrix followed by cyclic sequencing. The shift to single molecule PCR-free protocols and cycle-free chemistry is broadly characteristic of the progression to third-generation platforms.13 The advance of second- and third-generation technology has been enabled by innovation in sequencing chemistries, better imaging, microfabrication and information technology (IT). For the purpose of this review we will not discuss each platform in detail as these have been described extensively elsewhere.14,15 In addition, we will focus on the commercial second-generation platforms that are currently suitable for diagnostic applications, in preference to a detailed description of those platforms offered solely by sequencing service providers (Complete Genomics) or third-generation platforms such as Pacific Biosciences.16 Third-generation sequencing platforms offer many theoretical benefits relating to reduced cost, increased speed and removal of PCR-bias, however, the technology is still maturing and it is likely to be a few years yet before such platforms seriously rival the second-generation instruments and enter mainstream diagnostic use.
Second-Generation Sequencing Platforms
There are currently three companies offering second-generation sequencing platforms: Roche, Illumina and Life Technologies. Each company entered the market with large-scale instruments and maximum output in mind to satisfy a research market that demands a high-throughput technology for discovery-based applications and whole genome sequencing potential. Roche was the first to enter the market, acquiring the company 454 Life Sciences from its founder Jonathan Rothberg. The Roche 454 platform distinguishes itself from the other two large-scale platforms with longer read lengths, which are now approaching those of Sanger sequencing (700–1000 base pairs (bp)). The total sequence output from even the highest capacity 454 instrument (454 FLX+) is, however, far less than that of Illumina (HiSeq) and Life Technologies (SOLiD 5500), which generate many more sequence reads but of a much shorter length. Recently the attention has turned to smaller-scale low cost instruments with the introduction of the Roche 454 Junior, Life Technologies Ion Torrent and the soon to be released Illumina MiSeq, which are well suited to smaller research and diagnostic applications. A brief summary of currently available or near to release instruments and their performance is described in Table 1but we also refer the reader to a more comprehensive review of current sequencing platforms, their specifications and cost breakdown for further detail.17
Table 1.
Current platform options for second-generation sequencing.
Second-generation sequencers rely upon two principles: polymerase-based clonal replication of single DNA molecules spatially separated on a solid support matrix (bead or planar surface) and cyclic sequencing chemistries. Each platform is defined by the methods used to achieve these two processes. All platforms have similar front-end library preparation methods involving the addition of universal adapter sequences to the terminal ends of the DNA fragment. These oligonucleotide adapters are complementary to PCR primers used to amplify the library and oligonucleotides immobilised to a solid support for clonal DNA amplification. Both Roche (454) and Life Technologies (SOLiD 5500 and Ion Torrent) use emulsion PCR (emPCR) to generate clonal DNA fragments on beads.18 A water and oil emulsion is created where beads and template are added at a precise concentration such that each emulsion droplet is likely to contain a single bead and single DNA molecule. Following emPCR the emulsion is broken, the template carrying beads are enriched and then deposited into separate ‘pico-wells’ or bound to a derivatised glass flow cell.19,20Illumina uses an alternative strategy by creating DNA clusters directly on the flow cell by bridge PCR.21 From a practical perspective there are advantages and disadvantages to each approach. The process of emPCR is labour intensive although each company has developed automation to partially reduce the labour burden of this process. Cluster generation by bridge PCR has been fully automated and is therefore more streamlined. The MiSeq instrument, in fact, will require no user intervention from cluster generation to data analysis, which is highly attractive from a process point of view. The potential downside to Illumina bridge PCR is that the success of cluster generation is not known until sequencing has begun, an expensive exercise if cluster generation fails. From our experience, cluster generation is typically quite robust provided the sequencing libraries are of high quality and the concentration of the library is accurately measured by quantitative PCR.
Each of the available platforms uses different sequencing chemistries and methods for signal detection. All 454 platforms employ pyrosequencing, whereby chemiluminescent signal indicates base incorporation and the intensity of signal correlates to the number of bases incorporated through homopolymer reads.22 Ion Torrent uses a similar sequencing-by-synthesis (SBS) strategy but detects signal by the release of hydrogen ions resulting from the activity of DNA polymerase during nucleotide incorporation. In essence, the Ion Torrent chip is a very sensitive pH meter. Each ion chip contains millions of ion-sensitive field-effect transistor (ISFET) sensors that allow parallel detection of multiple sequencing reactions.23 There have been recent reports that Roche will adopt a similar detection method to Ion Torrent through a licence from the British company DNA Electronics, which would make the 454 and Ion Torrent platforms essentially identical.24 The virtues of semi-conductor technology are that the instrument, chips and reagents are very cheap to manufacture, the sequencing process is fast (although off-set by emPCR) and the system is scalable, although this may be somewhat restricted by the bead size used for emPCR.25 The SBS chemistry used by both 454 and Ion Torrent is also conducive to longer reads. Ion Torrent is currently restricted to fragments much shorter than that of Roche 454 but this will likely improve with future versions. Both have the common issue of homopolymer sequence errors manifesting as false insertions or deletions (indels). Whether fine-tuning the detection and analytical software can improve this issue is yet to be seen.
The Illumina and Life Technology SOLiD 5500 platforms are both considered short read sequencers but employ very different sequencing chemistries. Illumina uses reversible dye terminator SBS chemistry involving iterative cycles of single base incorporation, imaging and cleavage of the terminator chemistry. SOLiD uses sequencing by ligation (SBL) involving iterative rounds of oligo ligation extension, which is where the name originates (Sequencing by Oligo Ligation Detection). The principle of SBL in the context of massively parallel sequencing was originally described by Church and colleagues.26 The SBL process essentially measures every base twice by dinucleotide encoding, which is translated into ‘colour space’ rather than conventional base space. The new ‘Exact Call Chemistry’ offered with the 5500 line instruments uses a three base encoding, allowing even greater accuracy and actual base calls. Illumina SBS has a slight advantage over SOLiD SBL in terms of read length, now up to 100 bp on HiSeq and 150 bp with other Illumina instruments. The SOLiD SBL chemistry has a maximum read length of 75 bp but the two or three base encoding provides higher accuracy over the Illumina chemistry. Both Illumina and SOLiD platforms have a paired-end or mate-paired capability enabling reads to be generated from both ends of a single clonal fragment, as do the Roche 454 platforms.
Choosing a Platform
In choosing a platform there can be many considerations. Principally, one may be concerned with performance metrics such as read length, accuracy and total sequence output. In general, all second-generation platforms produce data of a similar accuracy (98–99.5%), relying upon adequate sequence depth or ‘coverage’ to make higher accuracy consensus base calls (>99.9% accuracy). Some systematic biases have been reported when comparing between NGS platforms and with other orthologous technologies such as Sanger sequencing. For example, significant non-uniformity of sequence coverage has been reported with short read instruments, whilst systematic errors have been reported for all NGS platforms.27–29 Systematic errors rather than random errors are more problematic as they cannot be overcome with higher read coverage. Modifications to protocols suggested by large genome centres may help to improve some uniformity issues,30 whilst newer read alignment and variant calling strategies have helped to reduce systematic errors caused by multi-mapping of reads or presence of indels (see Bioinformatics section below). Read length may be important for specific applications, such as identification of complex structural rearrangements or mapping across repetitive sequences. There is also some advantage in longer reads for direct amplicon sequencing.
Additional process-related considerations such as sequence output and running cost need to be balanced against speed and hardware costs. The larger more expensive instruments tend to have longer run times but generate orders of magnitude more data at a fraction of the price. Molecular barcoding or indexing of samples can be used with all platforms, allowing pooling or multiplexing of samples for high-throughput and translation of lower sequencing cost to targeted sequencing applications requiring a relatively small number of reads. Very high-throughput, however, is required to fill runs on the larger sequencing platforms in order to take advantage of the capacity and reduced cost, although platforms such as the SOLiD 5500 have more flexibility in this regard. If fast turnaround and flexibility is paramount then the smaller-scale instruments are likely to be preferable. Usability and reliability of the equipment are important and often cannot be ascertained from the company marketing the product. A large online NGS community has emerged with user forums, news and blog sites often being useful for getting first hand experience and early insights into new equipment and methods (Table 2). Informatics is also a very important consideration. Informatics hardware cost is largely dependent on the throughput of the sequencer. Larger sequencers require Linux servers with multiple cores and large amounts of RAM at a significant capital cost and require dedicated human resources to maintain, whilst smaller-scale sequencers can be run from high-powered Windows or Linux-based desktop systems.
Table 2.
Internet genomics news, forums and blog sites.
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Sequencing Applications
Whole Genome Sequencing
The predominant application of NGS in a clinical setting will undoubtedly be resequencing of genomic DNA. Whole genome sequencing (WGS) simply provides the ultimate genetic survey of an individual’s genome or cancer genome where a detailed map of single nucleotide variations (SNV), indels, complex structural rearrangements and copy number changes can be attained in a single assay.31 As the sequencing technology has advanced there have been major improvements to data quality and throughput, driving the cost of WGS towards $1000/genome, a threshold widely considered to be the tipping point for widespread clinical implementation. However, generating sufficient data for WGS remains a relatively expensive exercise for most laboratories. Strong competition between large service providers means that outsourcing WGS is currently a more affordable option for most and perhaps one that will persist until the third-generation technologies mature and become mainstream. An important consideration for sequencing whole genomes is that generating the actual sequence data is only a fraction of the total cost and does not take into account the expense associated with data storage, analysis and interpretation.32 Sequencing an entire genome reveals an enormous amount of genetic information, some of which can be interpreted and actionable, but a significant amount will be either novel and/or of unknown clinical importance. Additionally, there are significant ethical issues concerning privacy of data and incidental findings that will need to be resolved. For these reasons a more targeted approach to genome sequencing seems to be the logical first step towards widespread clinical implementation of the technology.
DNA Library Preparation
Regardless of the technology, all NGS platforms follow similar molecular protocols for the preparation of sequencing libraries. Although standard library preparation does not necessitate any specialised equipment, there are a number of auxiliary instruments that can aid in the library preparation process (Table 3). For the preparation of ‘shotgun’ fragments, DNA is sheared either mechanically or by enzymatic digestion to create fragment sizes in a required size range. A series of enzymatic steps are then used to repair library DNA ends and ligate common adapters that are complementary to oligonucleotides on beads or flow cells. Library preparation reagents are provided in kit form by the major NGS vendors and are also available through third-party companies. A relatively new alternative to the conventional fragment library preparation involves an in vitrotransposition method (Nextera, Epicentre/Illumina), which removes the need for mechanical shearing and multiple enzymatic and purification steps. PCR is commonly used to amplify libraries prior to sequencing, however, the number of cycles is often limited to avoid excess PCR duplicates that can contribute to false-positive sequencing error. The use of PCR and common adapter sequences introduces a high risk of cross contamination between libraries therefore standard principles of pre- and post-PCR work areas are essential. Size selection of DNA libraries may be necessary to aid in analysis and standardise cluster size. Traditionally, size selection has been done using either agarose or polyacrylamide gel electrophoresis (PAGE), however, new automated methods are also available. Finally, high-throughput library preparation can be automated and there are several commercial solutions currently available.
Table 3.
Auxiliary laboratory instrumentation for next-generation sequencing library preparation.
Sequencing Requirements
To overcome the higher error rate of NGS platforms compared to traditional Sanger sequencing a high level of redundancy or sequence coverage is required to accurately call bases. Typically, a 30–50x coverage is required for accurate base calling, although this can vary based on the accuracy of the sequencing platform, variant detection methods, and the material being sequenced.33 Using the Illumina HiSeq instrument approximately 100 Gb of sequence data is required to sequence a diploid genome or about three lanes of a flow cell using the new V3 sequencing reagents. Less coverage may be required on the SOLiD 5500 platforms owing to the higher read accuracy enabled by the two base encoding. Greater depth may be necessary for interrogating cancer genomes where normal tissue contamination and the heterogeneity of some cancers can reduce variant allele representation in sequence data well below the 50% frequency expected for a diploid heterozygous call.
Targeted enrichment before sequencing can reduce costs, allow higher coverage over regions of interest and potentially simplify the bioinformatic interpretation of NGS data. The amount of sequencing required for targeted applications will ultimately depend on the method and target region size. As an example, for whole exome sequencing (targeting all annotated coding genes) approximately 10–12 Gb of data is required, achieving an average of 100-fold coverage and at least 20-fold coverage for 80–90% of targeted bases. At current specification this means up to 32 exomes can be run per flow cell on the HiSeq instrument with similar throughput likely to be possible on SOLiD 5500. The smaller sequencing instruments such as MiSeq generate substantially less data (∼1 Gb) and therefore are suited to smaller targeted sequencing applications where, at most, a few hundred genes could be sequenced in a single run.
Targeted DNA Sequencing
Targeted enrichment strategies feeding into NGS are finding traction in both research and clinical diagnostic fields. An assortment of methods and technologies has been described in the literature, most of which can now be purchased as commercial products (Table 4). When comparing these approaches there are several factors that need to be considered. From a technical perspective the fidelity of capture is important. Off-target enrichment and low uniformity of capture can mean more sequencing is required to attain adequate sequence depth for all targeted regions. Different capture methods can also be affected by sample quality and the presence of variants within the capture region. Scalability, throughput and ease of use are important for high-throughput, whilst the targeted region size may dictate what method is most appropriate. Finally, the need for specialised equipment and the reagent price are also key considerations.
Table 4.
Targeted enrichment methods for next-generation sequencing.
Targeted enrichment methods fall broadly into two categories: PCR-amplicon and hybridisation capture approaches. As PCR-based approaches are already used routinely in diagnostic laboratories they fit well with existing diagnostic workflows. PCR is highly specific and has the advantage of generating more uniform coverage than comparative hybridisation approaches, provided the concentrations of individual PCR products are adequately normalised before pooling and sequencing. Different strategies have been used to generate PCR amplified libraries. Some use concatenation of PCR products to generate fragment libraries; shearing PCR concatamers and feeding into shotgun library preparation. A more straightforward protocol that is compatible with long-read sequencing instruments is to incorporate the sequence adaptors into the 5′ -end of the PCR primer enabling pooling of amplicons and direct sequencing. Conventional PCR methods are obviously better suited to targeting a small number of regions as the logistics, cost and amounts of DNA required to assay larger regions can be prohibitive.
Long-range PCR (LR-PCR) can reduce the burden of generating tens of PCR primer sets to amplify across regions of interest and has been employed to target contiguous regions or to amplify across several exonic regions. Uneven coverage can be an issue using LR-PCR although some remedies to this have been described.34 Generation of long amplicons can also be prone to reproducibility issues and is inherently not suited to the use of degraded DNA such as from formalin fixed paraffin embedded (FFPE) material. Additionally, the need to generate shotgun sequencing libraries post-PCR, regardless of sequencing platform, creates further work, expense and potential risk for failure and contamination.
The key to scaling PCR-based sequence enrichment involves automation, miniaturisation and multiplexing of PCR reactions. All of these methods aim to increase the scale of PCR enabling hundreds to thousands of PCR reactions whilst minimising reagent use, labour burden and amount of DNA template required. Two commercially available platforms enable miniaturised PCR by microfluidics. Fluidigm is a microfluidics-based method that uses multilayer soft lithography (MSL).35 A microfluidic circuitry is fabricated from a soft rubber composite that allows the controlled flow of reagents by using pressure to create tiny valves in the circuitry and reaction chambers for PCR. Fluidigm was originally developed for real-time quantitative PCR and single nucleotide polymorphism (SNP) genotyping applications but more recently the Access Array has been released, allowing retrieval of PCR product for targeted resequencing applications. The current Access Array system is capable of parallel PCR reactions for 48 samples by 48 single-plex assays. An attractive aspect of this platform is that relatively small quantities of template are required (∼50 ng/sample). Assays can also be multiplexed to improve throughput. Furthermore, as with many targeted approaches, index or barcoding tags can be incorporated into the universal adapter regions of the PCR product enabling the pooling of samples before direct sequencing.
A second platform, RainStorm (Raindance Technologies36), involves the generation of microdroplets in an oil emulsion, which then act as miniaturised reaction chambers for PCR.37 Highly uniform microdroplets containing reaction components (PCR primers and DNA template) are created using a combination of microfluidic chip design and high-pressure pumps. Thousands of reagent-laden microdroplets are loaded into a microfluidic device with a steady oil stream where they can be merged and manipulated through channels using electromagnetic fields before emPCR amplification and then retrieval of amplified product. Recently, this method has been marketed for the use of DNA extracted from FFPE tissue. The current limitations of this technology are the relatively large amount of DNA required and the sequential processing of individual samples.
The alternative to miniaturisation and microfluidic manipulation is to use methods enabling highly multiplexed PCR. One such approach uses molecular inversion probes (MIP). A MIP is a long oligonucleotide composed of sequence specific primer ends tethered by a universal linker sequence. Target specific primer ends hybridise to complementary DNA flanking the region of interest. Polymerase extension and then ligation results in the circularisation of the MIP. Captured regions are then amplified either by rolling circle amplification or by PCR from universal PCR priming sites within the linker region.38,39 The assay was originally described for amplification of exons targeting a relatively small number (10) of genes. However, the method was later shown to be highly scalable, and by using programmable microarrays, MIP pools targeting up to 50,000 exonic regions could be generated.40,41 The downsides to MIPs, however, are that they have been shown to provide inferior capture uniformity compared to hybridisation-based enrichment and they can be expensive for low throughput or custom applications as currently there are no commercial reagents available.
Illumina has developed a method that is similar in principle to MIP and is due for release in 2011 (personal communication, Illumina Australia). The ‘TruSeq Amplicon’ approach is derived from the method used for the Illumina ‘Golden Gate Genotyping’ assay. Instead of using MIP, two independent left and right flanking oligonucleotides are hybridised to a genomic DNA template enabling polymerase extension and ligation. Like MIP, the flanking oligonucleotides contain universal sequences for step-out PCR and incorporation of universal barcoded Illumina adapters. According to the vendor (Illumina) up to 384 targets can be amplified in a single reaction. The capture is theoretically very scalable, as all steps can be performed in a 96-well PCR plate and can be automated by liquid handling. The detailed performance specifications of this method are currently unknown.
Targeted enrichment by hybridisation capture has been extensively employed in a research setting and is typically suited for capture of larger target regions and exons from hundreds of genes. Oligonucleotides designed against complementary target regions are used as probes or ‘baits’ to hybridise and capture target DNA or ‘prey’ from pre-prepared shotgun libraries.42 The majority of methods employ microarray in situ oligonucleotide synthesis to generate the bait libraries (e.g. Agilent, Roche Nimblegen, Rivia), whilst Illumina uses its massive oligonucleotide production facilities to generate long oligonucleotides by conventional column-based synthesis. The microarray itself can be used as the capture device or the sequences can be cleaved from the array to generate in-solution bait libraries.43 The solution capture method has been more widely utilised owing to the scalability of the process, better performance for larger capture regions and no requirement for specialised equipment.44 The advantage of hybridisation enrichment is the ability to easily capture large regions in a single tube assay and it has become the mainstay of ‘exome’ enrichment resequencing for research.45–48 All companies now offer services for design of custom bait libraries in addition to off-the-shelf reagents for common applications, meaning fast turnaround and a price competitive market. The downside to hybridisation capture is the lack of specificity (compared to PCR) owing to cross hybridisation and lack of uniformity in capture, which relates to GC content of target sequence. Improvements to the method have been described, helping to overcome some of these issues, in addition to optimised methods for high throughput automation.49
RNA Sequencing
The application of RNA sequencing (RNA-seq) is fast superseding microarray technology by providing a superior digital readout whilst also enabling discovery of novel spliceforms, transcripts and RNA-editing.50 Quantitative gene expression data derived from RNA-seq has been shown to be comparable to that of microarrays but has better dynamic range and lower detection limit for lowly expressed transcripts.51Methods for analysing differential expression from RNA-seq data, however, are still being resolved and are reminiscent of the initial issues encountered for normalisation and statistical analysis of microarray data.52 Clinical applications of gene expression microarrays as a cancer diagnostic and prognostic tool have been demonstrated with examples including identification of tissue of origin for cancer of unknown primary and prediction of recurrence in early stage breast and colorectal cancer.53–55 Whilst it remains to be seen whether RNA-seq could replace microarrays or quantitative PCR as a clinical assay, it would seem intuitive to think that this could occur if the technology becomes cheaper and more robust.
RNA-seq has also been applied to mutation detection and has proven especially useful for the detection
