On July 6th 2017 was created the report on the extraction and quality control of my Aunt’s DNA. As written previously on March 22nd and April 19th, we are describing the process of sequencing the genome of my Aunt who passed away ~4 years ago (May 2013).
The purpose of this experiment is to perform a Direct-to-Consumer Whole Genome Sequencing of a deceased relative. This is to our knowledge the first study trying to achieve such a thing. We are interested to understand the extent to which the sequencing of relatives can inform the choices of the living.
An unexpected impact of this research is that I have received several contacts from people asking for help to sequence their deceased relative genome for sentimental reasons.
The ethical implications of sequencing people who are not today with us is a topic that remains to be explored. We are just scratching the surface. The fact that I have received several unexpected emails from people who want to do the same we are doing here is a clear indication that there is demand for sequencing the genomes of deceased relatives. Perhaps our DNA could be a better conduit for the preservation of our loved ones than other objects. Certainly it seems there are commercial opportunities here perhaps worth exploring.
I am very grateful for all the services that Mingo Bioinformatics is contributing towards the realisation of my goal to sequence my Aunt.
The results that I am going to write about below are very technical and I would not expect many readers of my blog to understand them. I add them here for the purposes of transparency and reproducibility. Our purified DNA sample has now been shipped to our sequencing provider in the US and a library of my Aunt’s DNA has been constructed. We are now only one step away from getting her whole genome sequenced and hence achieve this feat.
The plan is to make my Aunt’s DNA sequencing data freely available like the rest of my family. For me to be able to do this I have consulted with and requested permission from my Uncle to donate my Aunt’s DNA to the public domain (my Aunt’s husband and closest living relative – they did not have any children). My Aunt was included in the initial analyses of the Corpasome and she consented to have her 23andMe genotype openly shared with the world, hence, with all these cautionary measures, we believe we are in the situation to ethically be able to share her data in the public domain (CC0), like the rest of the family.
These are all the activities that have been carried out so far, which I will explain below for reproducibility purposes in great detail:
- DNA extraction
- Quantification and QA (spectrometry and fluorimetrics with Qubit®)
- Amplification via PCR with specific primers for a human gene (BRCA1) plus gel visualisation
- Library creation for DNA sequencing by US-based provider
Two samples of 7 hair roots of 0.5-1cm long were extracted on the 17th of April and 12th of May 2017. Both hair roots samples underwent exactly the same process and conditions for extraction. We used QIAmp® DNA Micro Kit Qiagen (#154044569) plus the QIAmp® DNA investigator protocol and a QIAcube automatic extraction equipment. The amount of the resulting solution was of 20ul.
DNA Quantification and Quality Control
We used ‘NanoVueTM plus’ to measure the concentration and purity of the DNA via a spectrophotometer. We used Qubit® to fluorimetrically quantify the DNA, using waves that selectively bind to the DNA to minimise contaminants.
Amplification via PCR and visualisation via agarose gel
For this we used New England Biolab (NEB): Q5 High fidelity DNA polymerase M0491S (#0051612) Deoxynucleitide (dNTP) Solution Mix N0447S (#0921703).
Our control sample (C+) was human DNA (100ng/l).
Our negative controlled was distilled water.
The preparation of the agarose gel (4%) and electrophoresis included agarose, a tampon Sybr safe DNA gel stain (Invitrogen) and a marker from Nippon genetics Eurpme. The load buffer used Promega Blue/orange 6x loading dye.
The volume of the PCR and loaded marker was of 3ul (plus a second load of the sample of 5ul)
Results of the Essay
(1) We consider these concentrations as the reference to perform the PCR assay
PCR and gel visualisation
The total amount of DNA for our sample id (id: 308) in the PCR reaction was:
- 7ul in the PCR reaction = 7 x 0.452 ng/ul = 3.2 ng in the reaction)
Dilutions of positive controls:
- C+_1: 100ng
- C+_2: 10ng
- C+_3: 1ng
Amount of time running: 20 minutes.
The visualisation of my aunt’s purified and amplified DNA (sample id: 308) compared with 3 DNA controls: 100, 10 and 1ng mass.
Library creation for DNA sequencing by US-based provider
Our DNA sample has now been shipped to our sequencing provider in the US. Our provider has now created a library and a QC for this library, producing this result:
The A1 lane corresponds to the QC test of my Aunt’s DNA.
We are now closer than ever to have the whole genome sequencing of my Aunt.
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Categories: Genome Diary