LIXIN: Intro My name is Lixin Chen, I am a research scientist
in the DNA Enzymes Division here at NEB and a member of the Evans lab.
Our recent publication in the journal Science is titled “DNA damage is a pervasive cause
of sequencing errors, directly confounding variant identification” and was performed
as a collaboration with Laurence Ettwiller, a Staff scientist and Pingfang Liu, a development
scientist here at NEB.
Segment 1
LAURENCE : This study has major implications for high throughput sequencing technologies
and the detection of somatic variants.
Somatic variants are sequences unique or shared with only a few cells in our body.
If somatic variants alter keys genes responsible for the control of cell proliferation, they
result in cancer.
During their growth, cancer cells acquire additional somatic variants leading to subpopulation
of cancer cells and tumor heterogeneity.
This subpopulation of cells can be resistant to otherwise working therapies and are therefore
responsible for the relapse of the cancer.
Thus, it is important to accurately identify all somatic mutations, even the ones that
are specific to only small subpopulation of cells.
PING : The development of high throughput sequencing technologies has revolutionized
our understanding of cancer biology and the treatment of cancer.
Sequencing of different tumor samples has identified many previously unknown somatic
variants, many of which have been successfully applied in targeted cancer therapies.
In addition, the ability to perform large scale sequencing of many tumor samples has
enhanced our view of tumor heterogeneity.
We now have a much better understanding of clonal selection and expansion, drug resistance,
and individual variation in response to targeted therapy.
The continued success in applying next generation sequencing to cancer therapy will largely
depend on the accurate identification of somatic variants of high sensitivity and high specificity.
Segment 2:
TOM : Sequencing accuracy directly impacts the identification of somatic variants.
Not surprisingly, DNA sequencing is not perfect and every so often mistakes occur.
These errors were usually ascribed to amplification mistakes or sequencing chemistry.
With modern high accuracy polymerases and sequencing chemistry other sources of error
can become problematic.
What makes this study so important is the discovery that DNA damage can be a prevalent
source of sequencing errors and fixing these damages greatly improves the detection of
true somatic variants.
Not all damage leads to sequencing errors, some lead to loss of signal while others are
more insidious, causing a polymerase to read the damaged base differently than the undamaged
base.
Guanine bases are known to react with oxygen forming 8-oxo-guanine which is read by many
polymerases as a T instead of a G.
This is insidious because unlike strand breaks where no information is generated, you instead
get incorrect data, which can look like a rare somatic variant.
Segment 3:
LIXIN : In this work, we identified DNA damage as a prevalent source of sequencing errors
by using two independent strategies.
The first strategy uses a DNA repair mix such as PreCR or the FFPE DNA repair mix to repair
the damage present in the DNA sample.
By sequencing the same DNA sample with and without the action of DNA repair, we can quantify
the level of damage present in a DNA sample.
LAURENCE : The second strategy uses the fact that a damage triggers the polymerase to almost
always responds with a similar behavior.
For example as tom mentioned, 8-oxo-guanine is misread by the polymerase as a thymine
(T) instead of a guanine (G).
Sequencing of 8-oxoG containing DNA will result in an imbalance between the rate of G to T
compared with the rate of C to A variant which is the reverse complement of G to T.
The difference between these two rates corresponds to damage.
Based on this principle, we computed a simple metric called GIV score to measure all damages
in the DNA and validated this metric using the experimental setup described by Lixin.
Segment 4: LAURENCE : The next question that we wanted
to address now is how prevalent is this damage in sequencing facilities that handle real
cancer samples ? We therefore requested access to The Cancer Genome Atlas (TCGA) initiative
data and found that more than 80% of the datasets have a significant imbalance of G to T indicative
of damage.
It is likely that the majority of sequencing currently done have large degrees of undetected
damages.
Consequently the rare and possibly interesting variants are confounded with incorrect data
derived from damage.
LIXIN : Closure: For more details about this work, you can read our publication in Science
in the Feb 17th issue, 2017.
This work is also available open access on biorxiv.
(http://biorxiv.org/content/early/2016/08/19/070334).