1. No category

TECHNICAL RESPONSE
◥
ANTHROPOLOGY
Response to Comment on “Late
Pleistocene human skeleton and
mtDNA link Paleoamericans and
modern Native Americans”
Brian M. Kemp,1* John Lindo,2 Deborah A. Bolnick,3
Ripan S. Malhi,2,4 James C. Chatters5*
Prüfer and Meyer raise concerns over the mitochondrial DNA (mtDNA) results we reported
for the Hoyo Negro individual, citing failure of a portion of these data to conform to their
expectations of ancient DNA (aDNA). Because damage patterns in aDNA vary, outright rejection
of our findings on this basis is unwarranted, especially in light of our other observations.
U
sing an analysis of nucleotide damage patterns and fragment sizes in Illumina sequences from the Hoyo Negro remains,
Prüfer and Meyer (1) suggest that the mitochondrial haplogroup D1 sequences we
obtained were not endogenous to the sample.
Although they raise important concerns, we note
that (i) the assignment of this individual to
haplogroup D1 was based on analyses of three
DNA extracts from two laboratories and (ii) expectations for the amount of damage observed in
an ancient sample of this age and context are not
firmly established.
We took precautions to minimize contamination from exogenous DNA [see the supplementary
materials (SM) in (2)] and conducted several analyses to assess whether the extracted DNA exhibited expected characteristics of ancient DNA (aDNA)
and to confirm haplogroup D1. We made the following observations: (i) this haplogroup was not
detected in negative controls; (ii) amplification
of mitochondrial DNA (mtDNA) fragments <200
base pairs (bp) was not uniformly successful (i.e.,
sporadic amplification was observed); (iii) failure
to generate X- and Y-chromosome amplicons; (iv)
AluI site loss at nucleotide position (np) 5176, diagnostic of haplogroup D, was confirmed through
intra- and interlaboratory replication using three
DNA extractions; and (v) hypervariable region sequences from all three extracts yielded differences
from the revised Cambridge Reference Sequence
(3) consistent with membership in haplogroup
1
Department of Anthropology and School of Biological
Sciences, Washington State University, Pullman, WA 99164,
USA. 2Department of Anthropology, University of Illinois,
Urbana, IL 61801, USA. 3Department of Anthropology and
Population Research Center, University of Texas at Austin,
Austin, TX 78712, USA. 4Institute for Genomic Biology,
University of Illinois, Urbana, IL 61801, USA. 5Applied
Paleoscience and DirectAMS, 10322 Northeast 190th Street,
Bothell, WA 98011, USA.
*Corresponding authors. E-mail: [email protected] (J.C.C.);
[email protected] (B.M.K.)
SCIENCE sciencemag.org
D1. Collectively, these observations are consistent with a low copy number and a highly degraded DNA sample, characteristics expected
from aDNA (4). Moreover, our results make phylogenetic sense (5).
As disclosed [SM in (2)], an attempt to reconstruct a complete mitogenome from the first
extract (HN-WSU-1) was compromised by crosscontamination from a sample (of haplogroup C)
processed in parallel at the University of Illinois.
Consequently, we used the high-throughput data
to confirm the presence of haplogroup D1 definitive single-nucleotide polymorphism (SNPs) [SM
(2)], similar to the approach of screening SNPs in
deeply sequenced amplicons compromised by contamination (6). We did not include an assessment
of postmortem damage in the SM for two reasons.
First, the DNA library was prepared using AToverhang adapter ligation and a proofreading
enzyme (Kapa HiFi DNA Polymerase), which
limits our ability to detect nucleotide misincorporation damage patterns (7). Second, because
most sequence reads did not contain SNPs specific to haplogroup D, it was not possible to
present a damage profile specific to the complete
D1 mitogenome.
Prüfer and Meyer’s (1) efforts to disentangle
sequence reads from multiple mitogenomes and
assess them for signs of DNA damage are informative. However, they considered only two diagnostic SNPs for haplogroup D1. We followed their
example and examined reads containing any of
the eight diagnostic SNPs for subhaplogroup D1
(nps 2092, 3010, 4883, 5178, 8414, 14668, 16325,
and 16362). Through this analysis, we identified
previously undetected polymerase chain reaction
(PCR) duplicates (i.e., reads that had been trimmed
to different lengths based on sequence quality).
Our analysis of the Illumina data set reveals only
27 unique reads with diagnostic haplogroup D
SNPs, sequenced to a 3.86× depth. Fragment
length ranges from 35 to 100 bp, with an average
of 85.8 bp. In these 27 reads, we see no clear indi-
cation of G-to-A misincorporations near the 3′
end of reads or C-to-T misincorporations near
the 5′ end, confirming the general observations
of Prüfer and Meyer (1).
Prüfer and Meyer (1) argue for a specific DNA
damage pattern as a reliable means of authenticating aDNA sequences, but that expectation
makes assumptions about the rate of deamination over time and the frequency of resulting
nucleotide misincorporations. Because this form
of damage is tremendously variable (8, 9) and
not time dependent (8, 10), it is unclear whether
postmortem damage at the 3′ and 5′ ends should
be observable among so few reads from Hoyo
Negro. If damage observed in reads from the
~30,000-year-old Kostenki 14 human (~38%) (9)
is an appropriate model for Hoyo Negro, the
binomial probability of observing 0/27 reads with
damage is 2.4 × 10−6. However, if we accept a
model of 1 to 9% damage (from Arctic samples
≤ 5400 years old) (8), the probability would be
0.078 or higher. It is probable that neither model
is appropriate for the Hoyo Negro remains. Thus,
although the absence of damage-induced nucleotide misincorporations in the Illumina haplogroup D sequence reads does raise concerns about
contamination, it is also possible that the 27 reads
showing diagnostic D SNPs are derived from endogenous aDNA and that we cannot detect postmortem damage due to chance and/or our library
preparation methods.
If the Hoyo Negro mtDNA results represent
contamination, its source is unknown. Because
DNA analysis was a key objective of the study,
extreme caution was used in sample recovery and
transport. Haplogroup D1 is also absent among
the collection and laboratory teams. Finally, all
samples were submerged in 6% sodium hypochlorite before extraction, a reliable means of
surface decontamination (11). The putative contamination could have originated from laboratory
reagents, but the reagents used by each laboratory
originated from separate lot numbers. Although
contamination could affect multiple lots, the probability of observing haplogroup D1 contamination
in three independent extracts at two laboratories
is low, though not zero.
Although it might never be possible to authenticate aDNA from humans with absolute certainty (1), investigators have offered a variety of
recommendations for properly conducting this
type of research [e.g., (4, 5, 12)]. Prüfer and Meyer’s
(1) suggestion is that the authentication of human
aDNA should hinge on the presence of hallmark
damage signatures (9, 13). However, it is premature
to rely so heavily on such damage patterns without
a greater appreciation of the variance expected
across samples from different environments. Furthermore, although decades of research have suggested that contaminant DNA will be more intact
than aDNA, recent work brings this assumption
into question (9) and demonstrates that contamination can also take on forms of damage expected
of aDNA (14). We, therefore, still regard independent replication as an important criterion for
authentication in human ancient DNA studies,
despite the recent trend of ignoring this criterion.
20 FEBRUARY 2015 • VOL 347 ISSUE 6224
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R ES E A RC H
R ES E A RC H | T E C H N I C A L RE S P O N S E
Nevertheless, with ongoing analysis of DNA from
the Hoyo Negro remains, we are paying closer
attention to patterns of DNA damage as an additional means of evaluating the authenticity of the
sequence data.
RE FE RENCES
1. K. Prüfer, M. Meyer, Science 347, 835 (2015).
2. J. C. Chatters et al., Science 344, 750–754
(2014).
835-b
20 FEBRUARY 2015 • VOL 347 ISSUE 6224
3. R. M. Andrews et al., Nat. Genet. 23, 147 (1999).
4. S. Pääbo et al., Annu. Rev. Genet. 38, 645–679
(2004).
5. A. Cooper, H. N. Poinar, Science 289, 1139b
(2000).
6. D. L. Jenkins et al., Science 337, 223–228 (2012).
7. A. Seguin-Orlando et al., PLOS ONE 8, e78575
(2013).
8. M. Raghavan et al., Science 345, 1255832 (2014).
9. J. Krause et al., Curr. Biol. 20, 231–236 (2010).
10. L. Orlando et al., Nature 499, 74–78 (2013).
11. J. L. Barta, C. Monroe, B. M. Kemp, Forensic Sci. Int. 231,
340–348 (2013).
12. M. T. P. Gilbert, H.-J. Bandelt, M. Hofreiter, I. Barnes, Trends
Ecol. Evol. 20, 541–544 (2005).
13. S. Sawyer, J. Krause, K. Guschanski, V. Savolainen, S. Pääbo,
PLOS ONE 7, e34131 (2012).
14. M. García-Garcerà et al., PLOS ONE 6, e24161
(2011).
26 September 2014; accepted 14 January 2015
10.1126/science.1261188
sciencemag.org SCIENCE
Response to Comment on ''Late Pleistocene human skeleton and
mtDNA link Paleoamericans and modern Native Americans''
Brian M. Kemp et al.
Science 347, 835 (2015);
DOI: 10.1126/science.1261188
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