Q_and_A _12639521Q0145.pdf

Beta.SAM Q & A RFQ 12639521Q0145 extended to 05/24/2021

Q_1 - Is this contract only for sanger DNA sequencing, or is there also next-generation sequencing involved?

A - Just sanger sequencing for this contract.

Q_2 - As for pricing schedule, can you please clarify scope of work for fragment analysis?

A - We will be performing the PCR with tagged end labeled primer (6-Fam or Vic most common so far. Not sure if may add another dye) which are used for microsatellite analysis.

In particular, we hope to estimate fragment size based on fragment migration compared to a known ladder.

To date, all fragment analysis has been performed on an ABI 3730XL DNA Analyzer. We hope that this or equivalent is being used. We have not validated our methods using other machines.

We will dilute (usually 1:10, but this can be adjusted as needed). We would send about 20 ul of the diluted sample (unless more is requested) for analysis, primers present (we do not clean the PCR product; all analysis has been done with primers present and should not be cleaned) with expected fragment lengths between 60bp and 300bp

We will need it run with a ladder for size selection (GS500 or equivalent provided by the contractor) and resulting files (FSA file) sent to us.

After we get the results and we analyze data using PeakScanner, we may request further dilution of the sample sent (in case of signal strength is to high (sometimes occur since some samples are done in multiplex)

Samples are sent in as either plate of various specimens (48 - 94 samples with 6 multiplexed panels is our most common usage) or as singles specimens needing the 6 multiplexed panels

Q_3 Should we perform from DNA extraction to data analysis?

A - No. We will be providing the sample as needed for analysis. In case of sequencing, We will provide the DNA template with primer mixed. Unless otherwise requested by company, our standard protocol has been to send 2.5 ul purified DNA template (~20 ng/ul), 5 ul 5 umol Primer, and 7.5 ul of water. After sequencing, data is sent to us and we analyze the data ourselves. Extra DNA is sent in case a repeat is needed.