2nd _Q_and_A_12639521Q0145.pdf

2ND Q & A 12639521Q0145

Q_1 Will the primers needed for the Sanger sequencing and fragment analysis be supplied, or is this something Gene by Gene would be required to supply?

A - For sequencing, we will be providing purified PCR product mixed with the primer. Unless you have a different preference, our standard protocol has been to submit

2.5 ul of purified PCR product (between 5 ng/ul to 50ng/ul), 2.5 ul of primer (5 nmol), and 10 ul of water for a total volume of 15 ul.

For fragment analysis, we will be providing labeled PCR product diluted 1:10 in a total volume of 20 ul. Standard size ladder will be provided by contractor. Fragment sizes will be between 60 bp to 350 bp. Since some of our samples are done in Multiplex, after getting results from first run, we may request a second run with the possibility we may need you to dilute the samples we sent you to get further read (in case one of the signals is two high to get an accurate read)

Q_2 Are all the samples going to be sequenced for the same thing (1 primer pair for the entire batch of samples), or will there be multiple targets (different targeted regions for each sample)

A - All samples will be premixed with the primer and purified PCR product. They will need to under go sequencing reactions for sangier sequencing and then loaded onto sequencing machine.

Q_3 Is the ask for us to provide the sequences to then be analyzed on your end, OR do we need to analyze the data and report any variants?

A - We will do the analysis the Raw data using Sequencher or Genious.