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Old 03-09-2016, 01:36 PM   #1
dhumpie
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Default How representative is Sanger Sequencing for NGS?

Hi All,

New member here. I had a dig around with google but was unable to come up with an answer. I have a quick question with regards to the traditional Sanger Sequencing versus Next Generation Sequencing. I want to get a feel for the percentage of rRNA reads going into NGS after knockingdown rRNA in my sample. Would doing it the traditional way (i.e. cloning, ligating, transforming, picking colonies for sequencing) by picking say 20 colonies off a plate and sequencing be a good estimate of the percentage of rRNA background in my sample, or would this introduce bias and I would be better off doing a trial run for NGS?

Your thoughs, suggestions would be gratly appreciated.

Cheers,
Darren
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Old 03-09-2016, 02:34 PM   #2
HESmith
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That's a lot of unnecessary work and, if you're talking Illumina libraries, the adapters constitute an inverted repeat that might be unstable in bacteria. I'd recommend qPCR of the library w/ rRNA primers vs. your favorite housekeeping gene(s) for comparison. You can even perform qRT-PCR of your sample before library prep to calculate the degree of rRNA depletion.
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Old 03-09-2016, 04:01 PM   #3
cmbetts
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Do you have any friends regularly running the sequencer you're prepping for? Making a trial library and spiking in a tiny amount to another run would be cheaper and more quantitative than cloning then miniprepping and sequencing 20 colonies.

I was researching rRNA depletion methods a few years ago, and ~50k reads from a MiSeq (0.2% of a v3 run) will give you a very exact measurement of your rRNA content. While you could get away with even less, I found that sequence crossover in demultiplexing makes the numbers sketchy <10k reads. Join the glorious ranks of run leachers. Given the throughput of the instruments, you can do tons of fun things with a sequencer's scraps if you design the right assay (and have access to colleagues who were going to run it anyways).
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Old 03-09-2016, 05:28 PM   #4
HESmith
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If you're going to try cmbetts' suggestion, be sure that your index sequence is different from your friend's libraries.
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Old 03-10-2016, 02:49 PM   #5
dhumpie
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Thank you all for your input. It is greatly appreciated!

Cheers,
Darren
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