Group Title: BMC Bioinformatics
Title: Accurate and fast methods to estimate the population mutation rate from error prone sequences
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Title: Accurate and fast methods to estimate the population mutation rate from error prone sequences
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Language: English
Creator: Knudsen, Bjarne
Miyamoto, Michael
Publisher: BMC Bioinformatics
Publication Date: 2009
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Abstract: BACKGROUND:The population mutation rate (?) remains one of the most fundamental parameters in genetics, ecology, and evolutionary biology. However, its accurate estimation can be seriously compromised when working with error prone data such as expressed sequence tags, low coverage draft sequences, and other such unfinished products. This study is premised on the simple idea that a random sequence error due to a chance accident during data collection or recording will be distributed within a population dataset as a singleton (i.e., as a polymorphic site where one sampled sequence exhibits a unique base relative to the common nucleotide of the others). Thus, one can avoid these random errors by ignoring the singletons within a dataset.RESULTS:This strategy is implemented under an infinite sites model that focuses on only the internal branches of the sample genealogy where a shared polymorphism can arise (i.e., a variable site where each alternative base is represented by at least two sequences). This approach is first used to derive independently the same new Watterson and Tajima estimators of ?, as recently reported by Achaz 1 for error prone sequences. It is then used to modify the recent, full, maximum-likelihood model of Knudsen and Miyamoto 2, which incorporates various factors for experimental error and design with those for coalescence and mutation. These new methods are all accurate and fast according to evolutionary simulations and analyses of a real complex population dataset for the California seahare.CONCLUSION:In light of these results, we recommend the use of these three new methods for the determination of ? from error prone sequences. In particular, we advocate the new maximum likelihood model as a starting point for the further development of more complex coalescent/mutation models that also account for experimental error and design.
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BMC Bioinformatics


Methodology article


Accurate and fast methods to estimate the population mutation
rate from error prone sequences
Bjarne Knudsen' and Michael M Miyamoto*2


Address: ICLC bio, 8200 Arhus N, Denmark and 2Departmentof Biology, Box 118525, Universityof Florida, Gainesville, Florida 32611-8525, USA
Email: Bjame Knudsen bknudsen@clcbio.com; Michael M Miyamoto* miyamoto@ufl.edu
* Corresponding author


Published: II August 2009
BMC Bioinformatics 2009, 10:247 doi:10.1186/1471-2105-10-247


Received: 25 February 2009
Accepted: II August 2009


This article is available from: http://www.biomedcentral.com/1471-2105/10/247
2009 Knudsen and Miyamoto; licensee BioMed Central Ltd.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.



Abstract
Background: The population mutation rate (0) remains one of the most fundamental parameters
in genetics, ecology, and evolutionary biology. However, its accurate estimation can be seriously
compromised when working with error prone data such as expressed sequence tags, low coverage
draft sequences, and other such unfinished products. This study is premised on the simple idea that
a random sequence error due to a chance accident during data collection or recording will be
distributed within a population dataset as a singleton (i.e., as a polymorphic site where one sampled
sequence exhibits a unique base relative to the common nucleotide of the others). Thus, one can
avoid these random errors by ignoring the singletons within a dataset.
Results: This strategy is implemented under an infinite sites model that focuses on only the
internal branches of the sample genealogy where a shared polymorphism can arise (i.e., a variable
site where each alternative base is represented by at least two sequences). This approach is first
used to derive independently the same new Watterson and Tajima estimators of 0, as recently
reported by Achaz [I] for error prone sequences. It is then used to modify the recent, full,
maximum-likelihood model of Knudsen and Miyamoto [2], which incorporates various factors for
experimental error and design with those for coalescence and mutation. These new methods are
all accurate and fast according to evolutionary simulations and analyses of a real complex
population dataset for the California seahare.
Conclusion: In light of these results, we recommend the use of these three new methods for the
determination of 0 from error prone sequences. In particular, we advocate the new maximum
likelihood model as a starting point for the further development of more complex coalescent/
mutation models that also account for experimental error and design.


Background
The population mutation rate (0) remains one of the most
fundamental parameters in genetics, ecology, and evolu-
tionary biology [3-5]. This interest in 0 derives from the
fact that this parameter measures the effective size (Ne)
and whole-locus mutation rate (ji) of a population, which
are of great importance in understanding its demography


and history. Specifically, 0 is a compound parameter that
is calculated as the product of 2pNjt (with p = 1 or 2 for
haploids and diploids, respectively). Correspondingly, a
number of alternative methods are available to estimate 0
from a population sample of allelic sequences [6-8]. These
alternative methods range from relatively simple sum-
mary statistics (moment methods) to full coalescent/


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0
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BMC Bioinformatics 2009, 10:247


mutation models. Indeed, the estimation of 0 remains
central to even the most complex coalescent/mutation
models that are otherwise concerned with the determina-
tion of other population genetic parameters (e.g., for
growth, migration, and recombination).

A population sample of sequences is obtained from inter-
breeding or potentially interbreeding individuals and is
therefore usually associated with a small number of muta-
tions [9-12]. Thus, when estimating 0 from a population
sample, sequence errors can pose a real problem, since
their numbers can begin to approach or even surpass
those for the mutations [13-19]. This problem becomes
particularly acute when working with error prone data
such as expressed sequence tags (EST), low coverage draft
sequences, and other such unfinished products [20,21].
For example, an error rate of one mistake per every 500
nucleotides (e.g., as for an EST dataset obtained from sin-
gle sequencing passes) will make a significant contribu-
tion to the observed variation among sequences that differ
because of mutations by <1 to 2%. If uncorrected, such
errors can lead to an inflated estimate of 0 and even erro-
neous conclusions about the biology of their population
[1,2,18,221.


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Many sequence errors arise as random accidents that
occur during the nucleic acid isolation, cloning/amplifica-
tion, sequencing, and recording phases of a DNA sequenc-
ing study [11,23,24]. As chance events that are rare (even
for error prone data), each of these random mistakes will
most likely be limited to a single sequence, rather than
repeated among two or more different ones within the
population sample [1,15]. Thus, these random errors will
most likely inflate the number of singletons within the
dataset (i.e., polymorphic positions where one sampled
sequence exhibits a unique base relative to the shared
nucleotide of the others) (Figure 1). In contrast, these rare
chance mistakes will make a much smaller contribution
to the shared polymorphisms (i.e., variable sites where
each alternative base is common to at least two different
sampled sequences).

This study relies on the simple premise that the random
mistakes of error prone sequences can be avoided by
ignoring the singletons within their population sample.
This strategy is first used to obtain independently the
same new Watterson [25] and Tajima [26] estimators of 0,
which were recently reported by Achaz [ 1] for error prone
sequences. This approach is then implemented in the
recent maximum likelihood (ML) model of Knudsen and


Alignment position
Sampled

sequence 1 2 3 4 5 6 7 8 9 10


I A C A G T C G A T C

II C T

III T G T

IV G T T

V G T T

Singleton (*) or shared + + *

polymorphism (+)


Figure I
Multiple sequence alignment for five hypothetical sequences as sampled from a single population. Periods in
sequences II-V refer to the same base as in I. The dash refers to a gap. Variable sites corresponding to singletons are marked
with asterisks, whereas those representing shared polymorphisms are denoted with pluses.



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Miyamoto [2], which incorporates various factors of
experimental error and design with those for coalescence
and mutation. By relying on only shared polymorphisms,
these three new approaches allow for more accurate esti-
mates of 0. However, this greater accuracy comes with a
cost as singletons due to actual mutations are ignored
along with those due to random errors. To assess this
tradeoff, the three new methods are tested against each
other and their original predecessors that count singletons
with evolutionary simulations and/or analyses of a real
population dataset for the California seahare (Aplysia cali-
fornica). These tests document that these new approaches
offer reliable and fast alternatives for the determination of
0 from error prone sequences.

Results and discussion
Three new methods for estimating 0 from error prone
sequences
Infinite sites model
As in their original versions, the new Watterson, Tajima,
and Knudsen/Miyamoto methods rely on the infinite sites
model to accommodate a neutral mutation process
[27,28]. The infinite sites model assumes that only a sin-
gle mutation can occur at any homologous position of the
population sample. Thus, each variable site will be repre-
sented by only two bases that subdivide the sampled
sequences into two non-overlapping subsets consisting of
those with the first nucleotide versus the remainder with
the second base. Correspondingly, each mutation will
map to a specific branch within the sample genealogy,
which thereby partitions the sequences into their two
non-overlapping subsets. For example, the shared poly-
morphism at position 3 in Figure 1 is attributable to a
unique mutation along the internal branch that partitions
sequences I and II from III, IV, and V.

As random errors are treated as rare chance events, they
are also modeled in this study along with the mutations
by the infinite sites process. Thus, only a single random
error or mutation is allowed at any site of the sampled
sequences. In turn, each random error is limited to a sin-
gle sequence (and therefore to a particular singleton) in
contrast to a mutation that can also result in a shared pol-
ymorphism (Figure 1). The reason is that random errors
arise during the experimental determination and record-
ing of individual sequences, whereas mutations occur at
specific points within the sample genealogy. Thus, a
mutation along an internal branch of the genealogy will
result in a new base that will be shared by two or more of
its descendant sequences.

New Watterson estimator (0'w)
Define Ti as the length of time (as scaled by N, genera-
tions) during which there are exactly i ancestors for n sam-
pled sequences. Standard coalescent theory tells us that:


T ~ EXp( )


2
E[T] i(i-1)


[4,29,30]. The expected total branch length of the geneal-
ogy for n sequences (as measured in units of scaled coales-
cent time) can now be calculated as:


E[L] = iE[Tly
i=2


n-1
2X1
1=1


Let n, be the observed number of segregating (polymor-
phic) sites in the dataset. Under the infinite sites model, n,
also counts the number of mutations, since each observed
variable site is attributable to a single mutation. The
expected number of mutations per locus per unit of
branch length is 0/2. Thus, an estimate of 0 can be
obtained as:


E[L]


[251.

A new Watterson estimator that avoids singletons (and
thereby random sequence errors, 0'w) can now be derived
from Equation (4) by adjusting both its numerator and
denominator. The numerator is easily adjusted by count-
ing only the shared polymorphic sites in the original data-
set (n'). Although more complicated, the denominator
can also be readily adjusted by including in its calculation
only the lengths of the internal branches where shared
polymorphisms can arise (see below).

Let us again consider a point in the genealogy where there
are exactly i ancestors for the n sampled sequences. Look-
ing forward in time, the probability that a particular
branch of the genealogy is not chosen for the next split
(leading to i + 1 lineages) is [(i 1)/i]. Thus, the probabil-
ity that this branch remains unbroken to the present is:


i-I i n-2
--X--X...-
i i+1 n-1


i-1
n-1l


By combining Equations (3) and (5), the total length of
the external branches where singletons can occur can now
be calculated as:


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E[LL}] = _, iE[Tl}= 2. (6)
i=2
Our Equation (6) is equivalent to equation (10) of Fu and
Li [31], apart from our use of different symbols and terms
and of time as scaled by N, generations (rather than gen-
erations alone). Thus, as previously noted by them, the
total length of the external branches where a singleton can
occur is independent of the original number of sampled
sequences. Fu and Li [31] also obtained the variance for
the total length of the external branches as their equation
(14).

In an asymmetrical genealogy with a basal split of one ver-
sus (n 1) sampled sequences, a mutation in the internal
branch leading to the common ancestor of the (n 1)
group will result in a singleton within the dataset (i.e., a
variable site where the single nonmember sequence
exhibits the unique base) (Figure 2). Thus, the length of
this (n 1) basal branch (as weighted by its probability of
occurrence) must also be accounted for in the adjustment


1/6


of the denominator for O'w. The weighted length of this (n
- 1) basal branch can be calculated as follows. First, the
chance of this branch is determined as the probability of
an (n 1) asymmetrical topology or equivalently as the
probability that either one of the two basal branches for
the genealogy remains unbroken to the present. Accord-
ing to Equation (5), the latter probability is 2 [1/(n 1)].
Second, the unweighted length of this branch is then cal-
culated as the length of the time interval T2, which is 1
[Equation (2)]. Thus, the weighted length of the (n 1)
basal branch is:


n-1


By combining Equations (4), (6), and (7), O'wcan now be
obtained as:


2n, n-2
0w = s[[LI- [ [Ln-l] i=2 .
E[L]-E[L1]-E[Ln_-]1 i=


1

3/2


1/6


(a)


(b)


Figure 2
Representative symmetrical (a) and asymmetrical (b) genealogies for four sampled sequences. In all, there are
six symmetrical and 12 asymmetrical labeled genealogies for these four sequences [8,45]. This figure illustrates how the shape
of a genealogy affects whether a mutation will lead to a singleton or shared polymorphism. This heterogeneity contributes to
the variance of 0 for the three new methods. This contribution is in addition to the heterogeneity of the genealogical branch
lengths and Poisson mutation process [4,5]. The diamond highlights the common ancestor of the (n I) basal group of the
asymmetrical genealogy. The dotted and thin solid lines mark the basal branch leading to this ancestor and the external
branches, respectively, where a mutation will result in a singleton. The thick solid lines denote the other internal branches
where a mutation will lead to a shared polymorphism. Expected branch lengths are given in units of scaled coalescent time next
to each internode (those of the symmetrical genealogy are specific for its particular labeled history). Although both genealogies
have an expected overall length of I 1/3, the total length of the internal branches where a shared polymorphism can arise is 7/3
for (a) but only 1/3 for (b).



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In words, 0'wignores the singletons (and thereby random
errors) in the original dataset, while restricting the total
branch length calculation to only those internal inter-
nodes where a shared polymorphism can arise.

Apart from our use of different symbols and terms for cal-
culating the denominator, our Equation (8) is equivalent
to equation (13) of Achaz [1]. Achaz [1] also derived his
equation B22 for the calculation of the associated variance
for n's (i.e., his Var [S_ l]).

New Tajima estimator (O'T)
Let zi denote the number of observed pairwise differences
between sampled sequences i and j (for i # j). Standard
coalescent theory tells us that the expected waiting time
for these two sequences to coalesce is ~ Ne generations.
Thus, the expected value of each ,ij is 0, whereas that for
their sum is:

n 2 n(n-1)(9)

i=1 j=i+l

Rearranging Equation (9) leads to:

n n
O n(-1) XXi (10)
n 'i=1 j=i+l

[26].

The Tajima and Watterson estimators have the same
expected value of 0, even though the former is based on
the total differences between each sampled sequence pair
whereas the latter is obtained from the total number of
segregating sites within the sample. Their different sum-
maries of the observed variation are the basis of Tajima's
D that tests for departures from the standard neutral
model [32] (see also [31,33,34]).

To avoid random errors, Equation (10) can be adjusted in
a manner similar to that used to modify Equation (4) of
Ow. First, let x'j count the number of observed pairwise
differences between sequences i andj after the removal of
all singletons from their dataset. Then, the expected
number of singletons can be calculated from Equations
(6) and (7) as the total length of the external branches and
(n 1) basal branch (as weighted by its probability of
occurrence) multiplied by 0/2 {i.e., [1 + (1/n 1)] 0}. The
removal of each singleton from the original dataset
reduces the expected sum in Equation (9) by (n 1). Com-
bining these results, we obtain:


n n

i=1 j i+1


i=1 j=i+l


n(n-1)
2


n)O n(n3)
2


(11)


Rearranging Equation (11) then leads to:


n 2 =
S n(n-3) i= ziIl
v 'i=l j=i+l


(12)


Our Equation (12) for O'ris equivalent to equation (15) of
Achaz [ 1 ], except for our use of different symbols and total
7', (rather than average t',) for the population sample.
Achaz [1] also derived his equation B23 for the calcula-
tion of the associated variance for average x'j [i.e., his
Var[7_nl]).

New Knudsen/Miyamoto model (0'KM)
Knudsen and Miyamoto ([2], hereafter referred to as
"KM") developed a full coalescent/mutation model that
accounts for three specific factors of experimental error
and design: (a) For random sequence errors, (b) For
unobserved polymorphisms due to missing data, and (c)
For the uncertain assignment of the multiple sequencing
reads for a diploid or polyploid individual to its two or
more homologues. Their KM model uses recursion to cal-
culate an exact probability of the population sample
under the standard Fisher [35] and Wright [36] model for
reproduction (hereafter, referred to as "FW") and the infi-
nite sites process for both mutations and random
sequence errors [37,38]. Their model relies on ML to esti-
mate 0 and the expected number of errors per full length
sequence (e).

At the heart of the KM model is equation (1) of Knudsen
and Miyamoto [2], which is reproduced here as:


P (S) = 2S .1isi i(S))
SS )+Oils i j>i:s J~s,

+ 0 S(S -1)+o1|s| I Pc( Si(S)).
i:o,>o
(13)
The parameters and factors of this equation are defined in
Table 1. As one works backwards in time, the probability
that the next observed event is a specific coalescence is
given by the first term in the top line before the double
sum. As indicated by this double sum, if indeed a coales-
cent event occurs, then it will happen between two sam-
pled and/or ancestral alleles with compatible (if not


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Table I: Definitions of the parameters and factors used in Equation (13) of the KM model

Parameter or factor Description


The number of segregating sites within the current set of sampled and/or ancestral sequences
Counts the number of "singletons" for sampled or ancestral sequence i. Here, "singleton" refers both to the derived
mutations of the shared polymorphisms for the sampled sequences as well as to those of the observed singletons (in the
strict sense) within the original dataset (Figure 3).
Probability of a,(S), which is the current set of sampled and/or ancestral sequences prior to a mutation in sequence i
Probability of P3,(S), which is the current set of sampled and/or ancestral sequences after the coalescence of combinable
sequences i and j (s,~- s,; see below)
Probability of S, which is the current ordered set of n sampled and/or ancestral sequences (SI, S2, ..., sn) during a particular
coalescent interval in the genealogy
Sampled and/or ancestral sequences i and j (where i : j)
Signifies that the available regions of sampled and/or ancestral sequences i and j are at least compatible and that the two are
therefore combinable (i.e., can coalesce)
Measures the relative degree to which sampled or ancestral sequence i is a complete or partial sequence. Thus, Z, s,
summarizes the total available length of all sampled and/or ancestral sequences during a particular coalescent interval.
Summarizes the current number of sampled and/or ancestral sequences during a particular coalescent interval in the
genealogy


identical) sequences. Such sequences are referred to as
combinable (si s1).

The first term in the bottom line before the single sum is
then for the probability that the next observed event is
instead a mutation. As indicated by this sum, if indeed a
mutation occurs, then it will happen to a sampled or
ancestral sequence with at least one "singleton" (1i > 0).
Here, the definition of a "singleton" is expanded to
include the derived mutations of the common ancestors
for the different groups of related sampled sequences as
well as those for the singletons (in the strict sense) of the
original dataset (Table 1). This expanded use of the term
acknowledges that a shared polymorphism under the infi-
nite sites model is due to a unique mutation within the
common ancestor of those sampled sequences sharing the
derived base (Figure 3). Thus, even though they result in
shared polymorphisms among the sampled sequences,
these derived mutations are ultimately counted as "single-
tons" as one works backwards in time. This expanded def-
inition of a "singleton" allows for the economical use of
7i1 alone to track the mutations of both the original shared
polymorphisms and singletons.

In the KM model, the available regions) of each sampled
sequence is summarized as a closed interval(s) that is
scored over the range of (0:1). The IsiI factor then quanti-
fies the total amount of sequence available for this sam-
pled allele. For example, the (0.2:1.0) score for the
leftmost (first) sampled sequence in Figure 4a indicates
that it is lacking the initial 20% of the full multiple align-
ment. Thus, Isil = 0.8 for this partial, leftmost, sampled
sequence. In turn, as one works backwards in time, the
closed intervals for the available regions of the sequences
for common ancestors are calculated as the union of the
known lengths for their two immediate descendants.


Thus, the closed interval of the common ancestor for the
two leftmost sampled sequences in Figure 4a is (0.2:1.0)
u (0.0:1.0) = (0.0:1.0), with Isi| = 1.0.

The purpose of Yi |si| in Equation (13) is to track the total
available length of all sampled and/or ancestral sequences
for the detection of mutations as one works backwards in
time. The corresponding use of |si| in the bottom line of
Equation (13) allows for the adjustment of a7i due to
unobserved polymorphisms resulting from missing data.

The KM model uses only Equation (13) when working
with error free sequences. In turn, this model also relies on
equation (4) of Knudsen and Miyamoto [2] when dealing
with error prone sequences. This additional equation of
the KM model assumes that the random errors are uni-
formly distributed along the sampled sequences and that
their total number is Poisson distributed with an intensity
of k= Pe sj|. The inclusion of Y |sl in the calculation of
k allows for the adjustment of this error rate to account for
incomplete sampled sequences.

In contrast to its predecessor, the KM' model uses only
Equation (13) when working with either error prone or
error free sequences. As for the new Watterson and Tajima
estimators, the KM' model operates by counting only
those internal branches of the genealogy where a muta-
tion will result in a shared polymorphism (Figure 4b). The
sampled sequences are rescored as unknowns with empty
intervals (0), as is the sequence of the common ancestor
for the (n 1) basal group of each asymmetrical genealogy
(Figure 2). Correspondingly, |sil is then reset to 0.0 for
these sequences. The KM' model ignores the external and
basal branches of the genealogy, where a mutation will
result in a singleton, by multiplying aCi for each sampled
and/or (n 1) ancestral sequence by Isil = 0.0 in Equation


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m,



Pc(U,(S))
Pc(P,(S))

Pc(S)

s,, Si


Is,

IS|


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T4


A A G G


A G G


(a)


(b)


Figure 3
Illustration of how a shared polymorphism among the sampled sequences becomes counted as a "singleton" as
one works backwards in time. In (a), the A/G bases for a shared polymorphism are shown for four sampled sequences. The
three coalescent time intervals of this asymmetrical genealogy (T,, with = 4, 3, or 2 sampled and/or ancestral sequences) are
labeled on the left. The solid bar marks the G to A mutation in the common ancestor of the two leftmost sampled sequences
(open dot), which results in the A/G shared polymorphism. The first event is the coalescence of the two leftmost sampled
sequences in T4 (a). This coalescence reduces the number of alleles to three as the two leftmost sampled sequences in T4 are
replaced by their common ancestor in T3 (b). In the process, their shared polymorphism is replaced by the "singleton" of their
common ancestor. This replacement is the basis of the expanded definition for a "singleton" (Table I).


(13). In this way, the KM' model discounts the singletons
in favor of the shared polymorphisms within the dataset
without the use of equation (4) and its associated param-
eters (e.g., k and e) of the original KM model.

For sampled sequences without missing data, the previous
explanation is complete as to how the KM' model corrects
for the branches of the genealogy where a mutation will
result in a singleton. However, for incomplete sampled
sequences, the "above and below" test of the KM' model
also becomes necessary to correct for the regions of each
common ancestral sequence where a mutation in its inter-
nal branch will lead to a singleton, rather than to a shared
polymorphism, because of this missing information (Fig-
ure 4b). In this test, "below" refers to those sampled
sequences that belong to the monophyletic group of the
common ancestor in question. "Above" then corresponds
to the remaining, more distantly related, sampled
sequences. In Figure 4b, the two leftmost sampled
sequences are direct descendants of the first, leftmost,
common ancestor, whereas the other three are not. Thus,
these two sampled sequences lie "below," whereas the
other three occur "above" the internal branch for this left-
most common ancestor.


The above and below test checks whether comparable
information for a region is known for at least two,
descendant, sampled sequences below and at least two,
more distantly related, sampled sequences above an inter-
nal branch in the genealogy. If not, then a mutation
within this region of the ancestral sequence that corre-
sponds to this internal branch will result in a false single-
ton within the original dataset. For example, a mutation
within the first 20% of the leftmost common ancestor in
Figure 4 will result in a false singleton of the second sam-
pled sequence rather than in a true shared polymorphism
of the first and second. The problem is that the leftmost
sampled sequence is missing comparable information for
the detection of this mutation as a shared change in the
leftmost common ancestor. The above and below test cor-
rects for such regions of the common ancestral sequences
during the rescoring of their closed intervals and s,|i. As
the first 20% of the leftmost common ancestor fails the
below half of this test, the KM' model disregards this
region during the recalculation of its closed interval as
(0:2:1.0) and |si = 0.8 (Figure 4b).





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KM
model


10.8 11.0 11.0 11.0
(0.2:1.0) (0.0:1.0) (0.0:1.0) (0.0:1.0)


0


0 0 0


1.0 T2


1.0 T3 3.0


1.0 T4

1.0 T5
(0.0:1.0)


KM'
model




0.0 T2 0.0






0.0 T3 1.8


0o.o0 T4

0.0 T5
0


Figure 4
Genealogy for five sampled sequences illustrating how closed intervals, Is,|, and Z,i|s,I are calculated by the KM
(a) and KM' (b) models. The closed and open segments of each bar denote the available versus missing or ignored regions
of its sequence as scored over the range of (0:1). s, are calculated from these scores (numbers next to branches) and Z, |sj is
then determined as their sum for each T, (values on the far right). In contrast, the KM' model (b) rescores the sampled
sequences and the (n I) basal ancestral sequence of this asymmetrical genealogy (the diamond) as 0. Thus, these sequences
make no contribution to Z, s,j as their s, = 0.0. The KM' model also ignores the first 20% of the sequence for the common
ancestor of the two leftmost sampled sequences. This region is missing from the leftmost sampled allele and thereby fails the
"below" half of the "above and below" test (see text).




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BMC Bioinformatics 2009, 10:247


Evolutionary simulations and A. californica dataset
Evolutionary simulations
To test the three new procedures, evolutionary simula-
tions were conducted according to standard methods [4].
Two hundred datasets apiece were simulated for eight or
16 sequences of length 500 from a single population
under the baseline conditions of the standard FW and
infinite sites models with 0 = 1, 2, 4, or 8. In addition to
these baseline conditions, sequence errors were intro-
duced as four or eight randomly placed changes among
the eight and 16 sequences, respectively, for an expected P
of 0.5. Estimates of 0 were then obtained for the 200 data-
sets of each tested combination with the FW model and
the original and new Watterson estimators, Tajima esti-
mators, and KM and KM' models.

As expected, the FW model and original Watterson and
Tajima estimators overestimate 0 when the datasets con-
tain random errors (Table 2). In these cases, the random
errors are counted as mutations, thereby inflating their
estimates of 0. Furthermore, these overestimations are
greater for the Watterson estimator than for the Tajima
estimator. The reason is that each singleton makes the
same contribution as a shared polymorphism to the
number of segregating sites (ns) in Equation (4) of Ow, but
a smaller one to the sum of the pairwise differences in
Equation (10) of 9. Specifically, each singleton is limited
in Equation (10) to the (n 1) pairwise comparisons of
the unique sequence to the (n 1) other sequences. Thus,
the Tajima estimator is less vulnerable than the Watterson


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estimator to random errors even though its vulnerability
is still significant.

In contrast, the KM model underestimates 0 when the
sequences are errorless (Table 2). Furthermore, this ten-
dency to underestimate 0 is also evident (although not sig-
nificant) when the sequences contain errors (simulations
A2, A4, B2, and B4; see also [2] for additional cases). In
these situations, mutations are sometimes counted as ran-
dom errors, thereby deflating their estimates of 0. In cases
where few to no random errors are expected (i.e., finished
sequences), one can first perform a likelihood ratio test to
determine if e = 0 [39]. If this null hypothesis cannot be
rejected, then the KM analysis should be restricted to only
Equation (13). However, if > 0 according to this likeli-
hood ratio test, then equation (4) of Knudsen and Miya-
moto [2] is also needed and the user of the KM model
must remain aware that her/his estimate of 0 most likely
includes some slight downward bias.

Unlike their original versions, the new Watterson estima-
tor, Tajima estimator, and KM' model all consistently
recover the true 0 in the simulations both with and with-
out errors (Table 2). Furthermore, these three methods
also avoid the tendency of the KM model to underesti-
mate 0 due to unnecessary or "greedy" parameters for ran-
dom errors. In particular, the reliance of the KM' model
on one less equation [(4)] and fewer parameters (e.g., X
and e) makes it much simpler and less prone to over-
parameterizations than its predecessor.


Table 2: Results of the evolutionary simulations


Evolutionary simulations


Coalescent/mutation models


Watterson and Tajima estimators


0 n s


Al: I
A2: I
A3: I
A4: I
Bl: 2
B2: 2
B3: 2
B4: 2
Cl: 4
C2: 4
C3: 4
C4:4
Dl: 8
D2: 8
D3: 8
D4: 8


0.96 0.10
3.49 0.23
1.02 0.10
5.10 0.28
1.92 0.17
4.38 0.26
2.06 0.16
6.19 0.33


0.76 0.10
0.90 0.14
0.84 0.09
0.99 0.12
1.51 0.17
1.80 0.20
1.78 0.15
1.88 0.17


1.10 0.15
0.96 0.15
1.01 0.12
1.02 0.13
1.80 0.25
1.92 0.26
2.02 0.19
1.92 0.19
4.05 0.44
3.89 0.46
4.00 0.3 I
3.82 0.31
7.88 0.71
7.90 0.85
7.82 0.54
8.00 0.58


0.98 0.10
2.63 0.15
1.00 0.09
3.43 0.16
1.92 0.18
3.44 0.19
2.04 0.16
4.32 0.20
4.27 0.35
5.62 0.33
4.12 0.28
6.23 0.27
7.75 0.49
9.67 0.63
8.08 0.49
10.73 0.52


1.12 0.16
0.94 0.15
0.99 0.1 I
1.03 0.13
1.81 0.25
1.91 0.26
2.04 0.20
1.92 0.19
4.27 0.53
4.09 0.53
4.21 0.38
3.87 0.35
7.87 0.78
8.38 1.03
8.20 0.72
8.31 0.71


1.02 0.12
2.04 0.13
0.97 0.10
2.01 0.13
1.89 0.20
2.91 0.20
2.09 0.19
2.95 0.20
4.31 0.39
5.12 0.38
4.24 0.36
4.84 0.32
7.78 0.55
9.22 0.73
8.26 0.69
9.57 0.68


These results are for the standard FW model (0F,) and the original and new Watterson estimators (Owand 90'), Tajima estimators (0Tand 0'T), and
KM and KM' models (09M and 09',). They are summarized as the means twice their standard deviations for 200 simulated datasets apiece for each
of the 16 different combinations of 0, n (number of sampled sequences), and s (Al to D4). Mean estimates that are significantly greater than or less
than the true 0 are highlighted in italics and boldface, respectively. No results are provided for the FW and KM models with 0 = 4 or 8 (lower left
corner), since these calculations are too computationally intensive (see text).


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1.10 0.15
0.95 0.16
0.96 0.12
1.01 0.13
1.82 0.26
1.92 0.25
2.10 0.22
1.96 0.22
4.32 0.54
4.13 0.54
4.28 0.41
3.86 0.38
7.85 0.77
8.39 1.03
8.32 0.82
8.60 0.81







BMC Bioinformatics 2009, 10:247


The mean estimates of 0 for the new Watterson estimator,
Tajima estimator, and KM' model are also generally asso-
ciated with greater standard deviations than those for
their original versions (Table 2). The proximal reason for
these increased standard deviations is that the elimination
of singletons results in the loss of some actual mutations
along with the random errors. However, this cost of the
three new methods appears to be relatively small, given
that their standard deviations are never twice as great as
those for their original versions (with these discrepancies
usually being much smaller). Indeed, the standard devia-
tions for the three new methods are less than or equal to
those of their counterparts in four cases (simulations A2,
A4, and B4 for O'w and A4 for O'rin Table 2). Perhaps more
surprising is that the standard deviations for the new Wat-
terson and Tajima estimators are comparable to those for
the KM' model, particularly when 0 = 1 or 2. These simi-
larities in their standard deviations are surprising given
that the KM' model is a full ML model.

As summary statistics, the original and new Watterson and
Tajima estimators are all computationally fast, requiring
much less than 1 CPU second on a 2.4 GHz Pentium 4
CPU to analyze each of the simulated datasets. More
importantly, the KM' model is much faster than the FW
and KM models as documented by its completed analyses
of the more complex datasets (simulations Cl to C4 and
D1 to D4 in Table 2). In contrast, the FW and KM models
fail to complete their analyses of these more complex
datasets due to time and memory constraints. The KM'
model is much faster than the FW and KM models,
because it relies on only shared polymorphic sites. Less
variable positions results in faster coalescences and fewer
choices as one works back through the coalescent/muta-
tion recursion of Equation (13).

Aplysia dataset
The A. californica dataset was the original motivating force
behind the development of the KM model [2]. Thus, this
real dataset was also analyzed with the KM' model to test
further its performance against that of its predecessor. This
dataset consists of 18 sequencing reads for six diploid
individuals from a laboratory population of the Califor-
nia seahare at the Laboratory for Marine Bioscience, Uni-
versity of Florida (LL Moroz and AB Kohn, unpublished
data). Three cloned inserts were sequenced from each
individual as a pair of single sequencing passes starting
from both ends of an internal segment of 1731 base pairs
for the protein-coding region of the nuclear FMRF gene.
These pairs of passes overlap in the middle for nine
sequences, but at most by only 58 bases. Thus, these 18
essentially single-pass sequences contain many random
errors and some missing data and their assignments to the
two homologues of each diploid remain uncertain (even
though their individual sources are known). Correspond-


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ingly, the KM analysis of this dataset with 44 singletons
and 10 shared polymorphisms required the full use of its
factors for experimental error and design (i.e., for random
errors, missing data, and uncertain homologue assign-
ments).

A full ML analysis of this complex dataset by the KM
model proved too time and memory consuming and a
two step procedure was therefore adopted instead
whereby the number of errors was first ML estimated fol-
lowed by the determination of 0 for this ML value [2]. This
heuristic approach resulted in an estimate of 0KM = 6.32
for this sample, which still took more than 12 hours to
complete on the same CPU as for the simulations (see
above). In contrast, a full ML analysis of this dataset by the
KM' model using the same factors for experimental design
is much faster as it took less than 1 hour on this CPU to
obtain a similar estimate of 'KM = 7.17. Correspondingly,
nucleotide diversity (7x) for this sample is calculated as
(7.17/1731 positions) = 0.0041 mutations per site. To
summarize, the KM and KM' models both support similar
estimates of 0 for this real dataset, but the latter (as in the
evolutionary simulations in Table 2) is much faster as
confirmed by its completed, full, ML analysis.

Conclusion
Expressed sequence tags, low coverage draft sequences,
and other such unfinished products are known to contain
random errors due to chance accidents during their data
collection and recording [1,11,21,23]. However, such
sequences often constitute the only available allelic infor-
mation for a population and methods to deal with their
random mistakes are therefore needed to obtain accurate
estimates of 0 from these error prone data. This study is
based on the simple premise that random sequence errors
are distributed as singletons. Thus, one can avoid the ran-
dom mistakes of error prone sequences by ignoring their
singletons in favor of their shared polymorphisms (Fig-
ures 2 and 4).

This strategy is implemented in the new Watterson esti-
mator, Tajima estimator, and KM' model. These new
methods are all accurate and fast according to their evolu-
tionary simulations and analysis of the real complex data-
set for A. californica (Table 2). These methods come with
the cost of increased standard deviations, but this price
appears small or even negligible compared to their advan-
tages of significantly improved accuracy and/or computa-
tional speed. Obviously, additional evolutionary
simulations and applications to real datasets are now
needed to evaluate more fully under what conditions the
removal of singletons is warranted in light of this tradeoff.
Nevertheless, the current successes with the new Watter-
son estimator, Tajima estimator, and KM' model support
our recommendation that these three methods be given


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BMC Bioinformatics 2009, 10:247


serious consideration when estimating 0 from error prone
sequences.

Unlike the Watterson and Tajima estimators that repre-
sent summary statistics, the KM and KM' models both
constitute full ML models that offer the framework for the
further incorporation of other experimental and popula-
tion genetic factors [2]. In particular, such further devel-
opments are encouraged for the KM' model in light of its
current successes in the evolutionary simulations and A.
californica analysis (Table 2). For example, biased errors
within a sample due to the systematic misreading of spe-
cific bases during DNA sequencing, the postmortem bio-
chemical degradation of ancient DNA, and/or other such
sources of error can be accommodated by a finite sites
process that allows for repeated mistakes as well as muta-
tions at the same sequence positions [14-16,401. Likewise,
additional factors to account for other population genetic
processes such as recombination (which is most likely the
most important parameter overlooked in this study of the
nuclear FMRF gene for A. californica) can be accommo-
dated by the use of ancestral recombination graphs [411.
Inevitably, these more complex versions of the KM' model
will require the use of sampling based procedures for their
implementation (e.g., Markov chain Monte Carlo approx-
imations), since the current use of direct ML evaluation
will remain practical for only the smaller datasets and
simpler models [14,42-441.

Authors' contributions
BK derived the equations, wrote the computer program
for the estimation of 0, and conducted the evolutionary
simulations. MMM provided biological interpretations
about the results. Both authors contributed to the design
and main ideas of this study, wrote and edited the manu-
script, and read and approved its final draft.

Acknowledgements
We thank Leonid L Moroz and Andrea B Kohn for the use of their unpub-
lished A. californica dataset; William Farmerie and Michele R Tennant for
their suggestions about this research; the Carlsberg Foundation for its fel-
lowship to BK; and the University of Florida for its support. A copy of our
computer program to estimate 06', Q'T, and 6O'M is available upon request
from BK.

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