Help a non-biologist with some basic background

[MGN]

Hi all. This is a shot in the dark, but I was wondering if anybody could help me understand broadly how preparing a sample for sequencing works, like everything that happens from petri-dish (or cheek-swab, fecal sample, etc...) to the point that it goes into the machine. I have frankly a million questions for anyone who can help. In fact, if anybody is willing to spend 30-45 minutes talking me through some of these by phone/skype, I could buy you something nice from your Amazon wishlist, for example.

For example, let's say I want to sequence the genome of Salmonella, serotype XYZ. I'm assuming that first I have to get my hands on a sample of that, but then what? Does it need to be cultured to a certain critical mass? Do I need to test that I've got the right serotype first? And once we have a reliable sample, what do we do then?

I've seen references to "purification" happening at this stage; what is involved in that? I've also read references to samples having to be so many ng/uL (I think those units are right...), so is the rest just water, or is it a specific solution?

And as for a specific example, I was looking at the JGI database for a species of algae, and someone had sequenced the DNA, and separately also the Transcriptome (ESTs) with 454 and just posted the raw SSF files. I want to ask for more information about what that contains but I don't know where to start and what a biologist who has used 454 (which is not me) should already know.

I know that's a lot of questions and obviously I have a lot to learn, but of course anything would be helpful, or even any resources to read. And if you want to let me ask you questions over the phone in exchange for a gift, send me an email through the board :-) Thanks in advance.

[mastal]

For sequenicng in general, I would recommend that you navigate to the Illumina website,
and read whatever documentation they have there about sample prep and library prep, especially anything related to the types of samples you are working with.

http://www.illumina.com/technology/next-generation-sequencing.html

As for 454 and sff files, I'm not sure how much support you will find on their web pages,
but they should also have brochures and documents about their platform and the sample prep kits that they sell.

http://sequencing.roche.com/

and lex nederbragt's blog has a good description of the sff file format:

https://contig.wordpress.com/2010/10/28/newbler-input-i-the-sff-file/

[MGN]

Awesome, thank you for this.

[Jessica_L]

I would also recommend looking at something like the Qiagen website to get a feel for things like DNA extraction kits.

The answer to some (most) of your other questions is "it depends". You kind of have to work backwards from your experimental question/answer: you figure out how many sequencing reads you might need to get sufficient confidence in the base calls of your salmonella genome-- depends on read length, size of genome and the read depth you decided on. From number of reads and experiment type (whole genome in this case) you figure out which prep kit will best meet your needs, scientific and otherwise. The sample prep kit determines how much DNA you need for input, which in turn guides what kind of yield you want from your DNA extraction kit (i.e. from Qiagen). For example, if you need micrograms of input material for the sample prep, you need to maximize yield from the DNA extraction, so you're definitely going to need to culture the hypothetical cells you obtained. For nanogram/picogram scale library preps, you aren't going to need as much DNA so you should be able to get away with needing fewer cells to put through DNA extraction.

Does that make sense?