3 Genomics Based Diagnosis

Transcript

Welcome to the third talk of this week, that is genomics based diagnostic methods. In the previous talk we have already mentioned about the genomics application in detection of and sequencing of SiRNAs. But we will be talking about more detail into the different genomic platforms that is based on high-throughput sequencing.

So genomic based sequencing is also known as NGS or next-generation sequencing technology. It is basically pyrosequencing or high-throughput sequencing, and it is revolutionizing the field of pathogen detection in a variety of plant samples. Unlike other molecular methods which require prior knowledge of sequence information on the pathogens NGS approach is unlimited making it possible to detect any known and novel pathogens in a single assay. So, this is the greatest advantage of this particular genomics assisted diagnosis program, where we do not require any prior knowledge on the microorganism that is actually causing the illness to the plant. So, this approach is not a rapid test but important for identification of unknown bacteria viruses and viroids, has no previous sequence data of the organism is needed. So, that is why it is we mostly use for those pathogens which are not previously reported or goods origin is not certain. No previous sequence data of organism and there is also no need to culture the organism only we know that so, far we were able to culture only 10% of the total bacteria that is present on this globe and approximately, 90 percent of the total bacteria which are still unculturable in nature. And, that is why we are not aware of those microorganisms in general but we can definitely sequence those microorganisms which are not culturable through this particular technology that is genomics assisted in NGS platforms. Discovery of new viruses/viroids and new hosts has increase very rapidly after the introduction of the NGS. So, application of NGS in diagnosis of plant pathogens let us go for a case study how it is helping new pathogens to be identified.

The advent of high-throughput sequencing has made it feasible to determine the genome sequence of the phytopathogenic bacteria Pseudomonas syringae pv. actinidiae in short we call it (Psa). This is the causal agent of an emerging pathogen that is bacterial canker of kiwifruit in New Zealand. In a very short period of time this particular pathogen started infecting all the kiwifruit orchards in New Zealand some eight to nine years back and it causes a severe alert severe problem to the New Zealand kiwifruit growers as well as in the New Zealand economy that based on these industries. The original outbreak strain was sequenced using a Roche 454 GS junior sequencing platform and within the first few weeks the outbreak of a draft genome sequence was assembled and the causal agent was diagnosed and the biovar was established so it was possible to identify the particular pathogen along with its biovar only through the technology of NGS. All other relevant technologies were able to identify it up to a certain level but certainly not up to the level of biovar and that is why NGS technologies has shown in this particular case that how it is important and how it is giving service to different nations. So proper management schedule could be adopted due to rapid diagnosis of the pathogen in New Zealand and therefore, a fruit industry or Kiwi orchards were saved from the pathogen because of the help of this NGS technology. There are different NGS platforms for example like 454, Illumina, PacBio, Illumina MiSeq and these different platforms provide different type of services for example 454 platforms gives a 1 GB equivalent per run then in case of Illumina HiSeq 2000 we receive less than 300 GB data per run in case of PacBio it is less than 500 MB per run and it is 8.5 GB/run in case of Illumina MiSeq. But the read lengths of all the platforms are different in 454 the read lengths are less than 900 basis whereas, Illumina it gives short reads of around 100 bases in case of PacBio it gives very long reads of 3,000 and more bases and whereas Illumina MiSeq sequence it also give short reads of around 100 bases. So, depending on the investigating as necessary we can go for different types of sequencing platforms for identification of on diagnosis of the pathogens concerned. This is another chart where it is giving a comparative features of different platforms again HiSeq 2500, HiSeq 2500 with high throughput as well as Rapid mode then MiSeq, PacBio RSII and you can see that read lengths vary drastically in different platforms and that is why their yield per lane is also different incase of different platforms.

NGS can help in identifying of known and unknown plant diseases within a day that is the beauty again of the particular NGS platforms that it takes so less of a time and if it is properly utilized within a single day one can come to an conclusion what type of microorganism or what type of pathogen is associated with the disease that is manifested in a particular crop species. With NGS one can find out within a day whether or not plant material is infected with pathogens.

As this technique captures both usual and unknown suspects it is a true breakthrough in genetic plant diagnostics. PCR technique detects patterns like fungi, viruses and bacteria by multiplying specific parts of their DNA. The disadvantage of this is that the search is highly selectively one base his/her assessment of which pathogen is likely to be present on certain symptoms and then one adapt the analysis accordingly. So in case of PCR one has to presume that probably this pathogen is infecting the plant and one will design primers accordingly to detect that particular pathogen whereas in case of NGS no such assumption is required as its sequence the entire genome whether from the pathogenic fungi bacteria or virus or from the plant and then it differentiate the genome or assemblies that is not of plant origin or that is not of the host plant so that it can then are compared with the NCBI database and then see that unknown sequences match with a another known pathogen and then it can be very easily identified on the basis of this NCBI search. Whereas, if it is search gives no result then again one can conclude that the sequence that is obtained from the possible pathogen associated with the host plant is of a new or unknown pathogen origin.

So billions of building blocks per sample is generated so, unlike old sequencing techniques, NGS maps billions of genetic sequences for an entire plant sample. One can scan billions of nucleotides, the building blocks of DNA, and the order of this nucleotide determines to which type they belong. So again NGS generates billions of database and this billion building blocks can be then assembled and then they can be searched on NCBI database to come out to a conclusion which is quite different from the other nucleic acid-based technology like PCR technology which search for a definite length site specific amplification of the primers and then it amplifies only a particular gene or segment of a gene for identification of the pathogen origin. This reveals that plant’s own sequences and allows us to see which divergent sequences are present in the sample. One can conduct his/her analysis using advanced software that have ‘fed’ with genetic information on plants and pathogens which enable us to quickly issue a reliable and definitive answer. So depending on the quality of the DNA sample that answer can be even provided within a day. So this is how this NGS platforms are helping.

The speed is a major advantage in a sector where time is money for instance if a quarantine organism is discovered in the shipment normally all other biological products in the shipments are detained. So this leads to a significant loss of value. So a quick conclusion is required and that quick conclusion can be achieved by using of NGS platforms that is, within a day or two were it can be established that whether which seed lots or which biological material is infected and which is which lot is not so, that it can be released from the shipyard and we can prevent the loss of its value. The same principle applies to cultivation the longer it takes to acquire results that are certain the longer one has to wait to combat the disease. So all parties in the chains right from the producers, importers to exporters they all benefit from the rapid diagnostics because it can give them considerable cost savings. So NGS platforms is helping both at commercial level and different growers and producers and even exporter level so that they are able to reduce their a loss because of probable occurrence of a new pathogen.

So tracking and tracing the NGS technology is also suitable for tracking and tracing. Many bacteria and viruses are transmitted by a plant material like seeds. Because of this material are transported on such a large scale however in some times it is difficult to trace the origin of an infection. So since this is again a difficult situation so NGS platforms can come into rescue in this particular situation as well it ascertains the sequence of a pathogen and that we can figure out information such as this occurs naturally in a country or not so once we are certain that okay this seed lot is infected but the pathogen is also present in the same country then there is no required of quarantine regulations to be taken into account. So this type of decision making process can be achieved by NGS platforms.

So what are the steps involved in NGS based diagnosis – it involves mostly the first step the collection of specimens, then convert conventional testing of the specimens then extraction of DNA and RNA followed by sequencing, and then finally through bioinformatic tools we can go for identification of the pathogen.

So with this we have come to an end of the today’s talk where, we have seen that NGS can help us to accurately and very correctly detect the biological of pathogen that is causing a disease in certain crops or that is present in a particular shipment and we can identify the disease-causing agent at even viable level within the shortest possible time. So that is the importance and significance of NGS platforms and that we have also seen what are the different and NGS platforms that are available. Currently, you provide services to growers and exporters. So in the next talk we’ll be talking about immunology and antibody based diagnosis technologies.

Thank you very much.