Hello from Dale Yuzuki

[DaleYuzuki]

Hello everyone, my name is Dale and I've been on the vendor side of NGS for several years now.

2003 - 2005: Illumina's home office (San Diego, CA US), BeadArray product manager (arrays, scanner, software, reagents)
2006-2008: NIH sales representative for Illumina (Bethesda, Maryland US)
2009: Southeast sales representative for RainDance Technologies
2010: Southeast sales representative for Life Technologies SOLiD (yes I know)
2011: Market Development Manager, Americas for Life Technologies Ion Torrent and 5500 / SOLiD NGS

I've been lurking here since 2008 and decided to participate in a more open way. Keep up the great work on this site!

[fastidiousme]

Hi Dale,

I am new to seqanswers, joined only yesterday! Your work profile looks pretty impressive. I joined a company called Sandor Proteomics Pvt Ltd last month. As far as I know, this company employs an array of techniques developed by Illumina. I hope you wouldn't mind if I contacted you in case I got stuck working on an Illumina platform!

Cheers,

Shalini

[DaleYuzuki]

Hi Shalini, glad to hear from you.

I'd be happy to offer my perspective on the Illumina technologies, having been there for almost six years, and am very familiar all of their genotyping, gene expression, and methylation arrays. (Okay, as part of my day-job nowadays, also very familiar with all their NGS offerings as well...)

Feel free to contact me directly at dale ((at)) yuzuki.org.

Dale

[fastidiousme]

Reduced Representation Library

Hi Dale,

Thank you for replying to my previous post. I wonder if you have an idea about constructing reduced representaion libraries (RRLs). I have been looking for methods to short list a meth-sensitive and a meth-insensitive restriction enzyme to be able to digest an entire plant genome and construct an RRL for the same.

I have read multiple research papers on how to construct RRLs but none of them mentions the ways to narrow down to one a suitable RE for whole genome digestion.

Well no pressure but it would be great if you could help in this regard! I am kind of desperate for answers here.

Thanks,

Shalini

[fastidiousme]

Sorry, I just remembered that you had mentioned your email id in your previous post. Will send you an email the next time. My email id is [email protected]

Shalini

[DaleYuzuki]

Reduced Representation Bisulfite Sequencing

Yes this would be better handled offline or in the Methylation forum.

But I'll take a crack at it anyway here in the 'Introductions' forum for grins.

Due to the polyploidy of many plants doing RRBS is going to be tricky at best. Looking into the literature briefly that references the Meissner Nature paper from 2008, subsequent work was all done on 'mammalian genomes', with a particular emphasis on stem cells and cancer (predictably). The 'tricky' part comes in with the polyploid nature pf plants: until we get Oxford Nanopore 100K reads determining haplotype across the genome of a polyploid organism, much less a quantitative methylation status, that work is just going to have to wait.

Thinking about it further 'someone' could do many many PacBio runs to that same end, yet it would also be fraught with difficulties I can think of off the top of my head, not to mention very expen$ive.

So feel free to contact me directly with further discussion, I look forward to hearing from you.

Dale

[miroslav]

amplicon sequencing

Hi Dale,
Seeing that you've dealt with RainDance Tech previously I am wondering if you could answer the following question:
How is non-redundancy handled with amplicon sequencing?

Alternatively, if you could suggest where to look for answer that would be really appreciated too.
happy to be contacted directly on [email protected]

Thanks
Miroslav

[DaleYuzuki]

Hi Miroslav,

I'm not sure what you mean by 'non-redundancy' - could you please elaborate?

For what it's worth, each pico-liter-scale droplet has much less than one human genome equivalent in it. If I remember correctly it was on the order of 1/5th of a genome, so only one out of five droplets would actually produce a PCR product, and four out of five are 'empty'.

Look forward to hearing back from you.

Sincerely,
Dale

[miroslav]

non-redundancy

Hi Dale,
Thanks for your response.
Unfortunately I am unable to answer your question about the meaning of the term "non-redundancy". The questions popped up in a reviewer response without any context. My only thought was that it referred to representation of "difficult" regions that may not amplify sufficiently prior to sequencing.
Perhaps this was a writing mistake and the question meant to be about something else :-(
Regards
Miroslav

[DaleYuzuki]

Hi Miroslav, well let's just assume then that the term intended was 'redundant' regions of the genome in terms of amplicon specificity, which could be repetitive regions, pseudo-genes, or paralogous genes.

With a hybridization-based method (i.e. Agilent's SureSelect or NimbleGen's EZ Cap) these regions are simply 'repeat-masked' and designs simply cannot be made to those regions.

With RainDance, one of the nice things about it is that it is regular PCR primer design; a program like Primer 3 can be (and often is) used for the design, so there is a measureable increase in the percentage of successful designs. That being said, if you talk with RainDance customers they will tell you that the first exon of any gene is often not amplified in their enrichment (or not amplified very well) and this is a problem.

Hope this helps, feel free to ask further questions if you need any clarification.

Dale