Today's Question:
What are the current challenges in the field?
Dr. Crowe's answer:
Well, the technology is very powerful in that the numbers of throughput that can be achieved in the sequencing are impressive. But, the technologies are evolving constantly. There are a number of commercial suppliers of instruments and chemistries and re-agents and these are changing every couple of months. So, currently people are grappling with what are the best chemistries and how to deal with errors. So, all of these technologies involve polymerases that create errors and how to tell the difference between a sequencing error and a naturally occurring somatic variation is a big challenge in the field.
Also, the high throughput technologies give very good throughput, but relatively short read lengths. Currently, if you can get both ends of DNA to read 300 nucleotides in and stitch together and achieve something like a 500 nucleotide read, that’s considered a good effort. But, we need longer reads to sequence entire antibodies.
It is also very expensive right now. The price undoubtedly will come down and has been coming down. But it is still a relatively expensive technology and because of that, that has led people to try and combine samples. So, multiple samples in a single lane for sequencing to reduce the cost. This is multiplexing and the results can be deconvoluted if each of the amplicons is labeled in some way with a bar code or an index that is put on either with an adapter or a PCR. Learning which adapters and indexes and bar codes are compatible with various PCR primers is a technical challenge and deconvoluting the multiplex data set is somewhat challenging.
Another major obstacle in the field is that the numbers of sequences that come back are greater than the capacity of the software programs that we have to analyze antibody sequences. So, there are some great programs in the field – Immunogenetics Database (IMGT). There is another program from the NIH – JOINSOLVER and another program SoDA. All of these are from academic sources that are open, but they do not have the capacity to deal with millions or billions of sequences, for instance. IMGT currently has a high throughput upload of about 150,000, which sounds like a large number. But if you have a billion sequences, that really isn’t adequate to do your analysis. So, I think many people are scrambling in their own institutions or companies to develop local, proprietary methods for analysis and it’s not clear the best way to do that.
I think finally the biggest next step in this sequencing technology is to figure out a way to link the heavy and light chain sequences because antibodies are encoded by two different, major, recombined chains– heavy and light chain and, of course, the specificity antibody derived from both heavy and light chains together. But the sequencing separates heavy and light chains and if you don’t have the natural pairing, you lose a lot of information that is very important. So, I think an obstacle in the field is the technical difficult in molecularly linking heavy and light chains from single cells and then being able to sequence both of those chains together. There are very many challenges despite the exciting promises in technology.
Dr. Crowe will be presenting Deep Sequencing the Human Antibody Response to Viral Infections and Human Germline Antibody Gene Segments Encode Polyspecific Antibodies. For more information on these sessions and the rest of the program, download the agenda. The Antibody Engineering & Therapeutics Event will take place December 8-12, 2013 in Huntington Beach, California. If you'd like to join us, as a reader of this blog,when you register to join us and mention priority code XD13172BLOGJP to save 20% off the standard rate.
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