Seqanswers Leaderboard Ad

Collapse

Announcement

Collapse
No announcement yet.
X
 
  • Filter
  • Time
  • Show
Clear All
new posts

  • Help a non-biologist with some basic background

    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.

  • #2
    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.

    Discover the broad range of experiments you can perform with next-generation sequencing, and find out how Illumina NGS works.


    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.

    Sample prep for next generation sequencing (NGS) holds the key to unlocking the potential of every sample. Explore our integrated Roche Sample Preparation Solutions, encompassing all the steps required to convert a sample to a sequencing-ready library. From sample collection to library quantification, we offer sample prep solutions for different sample types and sequencing applications that are proven, simple, and complete. MC-US-09181


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

    Newbler can obviously take in the 454 reads, but also other read types: regular Sanger reads, any sequence in a fasta file (at most 200 bp), and perhaps also Illumina reads. Sff files are the stand…
    Last edited by mastal; 12-04-2015, 08:35 AM.

    Comment


    • #3
      Awesome, thank you for this.

      Comment


      • #4
        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?

        Comment

        Latest Articles

        Collapse

        • seqadmin
          Strategies for Sequencing Challenging Samples
          by seqadmin


          Despite advancements in sequencing platforms and related sample preparation technologies, certain sample types continue to present significant challenges that can compromise sequencing results. Pedro Echave, Senior Manager of the Global Business Segment at Revvity, explained that the success of a sequencing experiment ultimately depends on the amount and integrity of the nucleic acid template (RNA or DNA) obtained from a sample. “The better the quality of the nucleic acid isolated...
          03-22-2024, 06:39 AM
        • seqadmin
          Techniques and Challenges in Conservation Genomics
          by seqadmin



          The field of conservation genomics centers on applying genomics technologies in support of conservation efforts and the preservation of biodiversity. This article features interviews with two researchers who showcase their innovative work and highlight the current state and future of conservation genomics.

          Avian Conservation
          Matthew DeSaix, a recent doctoral graduate from Kristen Ruegg’s lab at The University of Colorado, shared that most of his research...
          03-08-2024, 10:41 AM

        ad_right_rmr

        Collapse

        News

        Collapse

        Topics Statistics Last Post
        Started by seqadmin, Yesterday, 06:37 PM
        0 responses
        8 views
        0 likes
        Last Post seqadmin  
        Started by seqadmin, Yesterday, 06:07 PM
        0 responses
        8 views
        0 likes
        Last Post seqadmin  
        Started by seqadmin, 03-22-2024, 10:03 AM
        0 responses
        49 views
        0 likes
        Last Post seqadmin  
        Started by seqadmin, 03-21-2024, 07:32 AM
        0 responses
        67 views
        0 likes
        Last Post seqadmin  
        Working...
        X