MiSeq workflow - tissue to instrument
Our lab is new to NGS and we are in the midst of planning our first sequencing run. Our project involves targeted resequencing of 15 horse genes, about 50 kb each. We are hoping to sequence the genes from ~ 100 horses. I'm trying to work through the procedure for doing this, but the plan I am coming up with seems prohibitively expensive. I am wondering if I'm missing something obvious, doing something wrong, or perhaps not approaching it in the best possible way. I would very much appreciate some feedback from those who know better!
Here's the basic workflow:
1. Harvest tissue from horse
2. Isolate genomic DNA
3. Using long range PCR (using, for e.g., SequelPrep, lifetech) amplify our DNA of interest in approximately 10 kb overlapping fragments.
Does this seem like an unreasonable workflow? Any alternative approaches? The PCRs and the cleanup alone will cost upwards of $20-30,000.
This is my first post, so if I've left out any info that might make it easier for you to answer, please let me know. I appreciate your responses!
Here's the basic workflow:
1. Harvest tissue from horse
2. Isolate genomic DNA
3. Using long range PCR (using, for e.g., SequelPrep, lifetech) amplify our DNA of interest in approximately 10 kb overlapping fragments.
-for 15 genes covering 50 kb each that comes out to ~ 75 PCRs / animal.
4. Clean up PCR reaction, quantify the amplicon-Using something like QIAquick PCR purification kit
-Quantify using nanodrop or Qubit (I think we'll be buying one)
5. Pool equal amounts of amplicon from each reaction into one sample / horse-Quantify using nanodrop or Qubit (I think we'll be buying one)
-i.e. One sample will now contain 75 different amplicons, in roughly equal amounts to ensure representative coverage.
-Repeat steps 2-5 for 96 horses, resulting in 96 samples, each with 1ng (or 50ng if we use nextera) of DNA total containing our target sequences, for library prep
6. Proceed with Nextera or Nextera XT for library prep-Repeat steps 2-5 for 96 horses, resulting in 96 samples, each with 1ng (or 50ng if we use nextera) of DNA total containing our target sequences, for library prep
-are there alternatives for tagmentation + indexing?
7. MiSeqDoes this seem like an unreasonable workflow? Any alternative approaches? The PCRs and the cleanup alone will cost upwards of $20-30,000.
This is my first post, so if I've left out any info that might make it easier for you to answer, please let me know. I appreciate your responses!
Comment