Hey guys-
As my username indicates, I don't have a lot of familiarity with NGS techniques. I've been trying to get advice from people here at my local institution about a project we want to do (described below) and have been getting different answers from different people.
Essentially, what we want to do is accurately quantify HIV progression through various stages of its life cycle. HIV is an RNA virus that converts its RNA into DNA (reverse transcription) through a complex process that involves DNA synthesis, RNA degradation, multiple strand transfers, etc. It is possible to design specific primers that can detect many of the intermediates (early RT, early-mid RT, mid-late RT, late RT). We can also design primers that detect the next step of the virus life cycle (integration) which can be productive (integrating into the host chromosome) or abortive (two types of auto-integrants called 1-LTR and 2-LTR circles). All of these things can be resolved by specific PCR primers except for integration into the host chromosome which usually depends on a virus-specific primer and a random primer since where the virus will integrate is not known.
What we want to do is get very accurate measurement of how many cells that are infected with HIV make it to the early RT stage, to the early-mid, mid-late, and late RT stages, and how many of those integrate correctly into the host chromosome and how many form 1- and 2-LTR circles.
To do this, we will need different primers and would be producing PCRs of various lengths for each step we are measuring. I am concerned that differences in primer-binding efficiencies and PCR product generation due to product length is going to introduce variability during amplification that will make it extremely difficult to accurately calculate the starting frequency (or at least the relative frequency of cells that have made it through the RT and integration steps).
One of the people I've talked to here is adamant about using NGS as the preferred platform and thinks we'll get more accurate quantitation as well as be able to determine that we are amplifying the correct products; the other is recommending digital droplet PCR and claims it will be more sensitive in our case because amplifying within droplets will allow us to accurately calculate starting frequencies even when there is some variation in amplification occurring.
Any advice from the sequencing community? Sorry the post is long but I could really use some advice.
cheers,
john
As my username indicates, I don't have a lot of familiarity with NGS techniques. I've been trying to get advice from people here at my local institution about a project we want to do (described below) and have been getting different answers from different people.
Essentially, what we want to do is accurately quantify HIV progression through various stages of its life cycle. HIV is an RNA virus that converts its RNA into DNA (reverse transcription) through a complex process that involves DNA synthesis, RNA degradation, multiple strand transfers, etc. It is possible to design specific primers that can detect many of the intermediates (early RT, early-mid RT, mid-late RT, late RT). We can also design primers that detect the next step of the virus life cycle (integration) which can be productive (integrating into the host chromosome) or abortive (two types of auto-integrants called 1-LTR and 2-LTR circles). All of these things can be resolved by specific PCR primers except for integration into the host chromosome which usually depends on a virus-specific primer and a random primer since where the virus will integrate is not known.
What we want to do is get very accurate measurement of how many cells that are infected with HIV make it to the early RT stage, to the early-mid, mid-late, and late RT stages, and how many of those integrate correctly into the host chromosome and how many form 1- and 2-LTR circles.
To do this, we will need different primers and would be producing PCRs of various lengths for each step we are measuring. I am concerned that differences in primer-binding efficiencies and PCR product generation due to product length is going to introduce variability during amplification that will make it extremely difficult to accurately calculate the starting frequency (or at least the relative frequency of cells that have made it through the RT and integration steps).
One of the people I've talked to here is adamant about using NGS as the preferred platform and thinks we'll get more accurate quantitation as well as be able to determine that we are amplifying the correct products; the other is recommending digital droplet PCR and claims it will be more sensitive in our case because amplifying within droplets will allow us to accurately calculate starting frequencies even when there is some variation in amplification occurring.
Any advice from the sequencing community? Sorry the post is long but I could really use some advice.
cheers,
john