Newbie question, terminology understanding for WGS...

[newkid]

Complete Newbie, and a current undergrad. I'm still trying to grip this entire field along with the vocabulary associated with it. So if my questions are a bit convoluted--I really appreciate your patience!

This is purely hypothetical, but....

....I'm sequencing a whole human genome from Saliva.


After taking the saliva sample the next step is sample prep (correct?)

Sample prep includes DNA amplification and purification (correct?)

After that, what are the samples loaded onto? I read that certain companies process 96 well plates (http://www.operon.com/services/dna-s...rice-list.aspx)

...so does that mean one sample per well? Or does one sample get divided into all 96 wells?

Then they're run through next gen sequencers? (Is there another process in between?)

After they're run through the sequencers, they go through bioinformatics processing. I get a fastq file with a bunch of short reads that have to be assembled (de novo or from a reference). [I have a pretty decent understanding of this area].


Or am I missing a whole lot? As you can see, it's really difficult for me to complete a picture without understanding the technical vocabulary.

[newkid]

Additionally, what's the most expensive part of this entire process? How does it make sense that companies can charge upwards of $2000 dollars for whole genome sequencing?

[CPCantalapiedra]

mmm I recommend you to read first some reviews about the topic, as well as download Illumina, SOLiD and Roche docs about their sequencing technologies and then the docs about the sample and library preparation.

But, if what you want it is a general picture, although I am pretty new to this field too, here it goes:

Saliva sample -> get rid of everything that is not DNA -> amplify DNA to obtain much more quantity so that further steps are able to work with it -> DNA quality check -> library preparation -> Sequencing Run -> Data Cleanup and filtering -> Data pre-processing -> Data Analysis

I guess that the most expensive step could be the Sequencing Run. However, the work with the data can be very expensive depending on the complexity of the experiment, if you take into account the people involved, the resources used (clusters, cloud time, high-memory front-ends, ...), and the time invested.

Library preparation is different depending on the platform you use, but as on your experimental design. It is not the same to sequence just the whole DNA from one sample, several samples you want to differenciate, ...

The sequencing run changes a lot too in technology, timings, etc depending on the platform.

Finally, the data analysis involves often to work with FASTQ files. However, 454 raw format are SFF files and there are a lot of formats involved in the process, like SAM/BAM format, mostly use as format for mappings (alignments); GFF, BED, etc.

Hope this helps, from a newbie to another one
Regards