# Evolutionary placement of short sequence reads on multi-core architectures.

**ABSTRACT** The application of high performance computing methods in bioinformatics becomes increasingly important because of the masses of data generated by novel short-read DNA sequencers. One important application of such short reads, is the analysis of microbial communities where the anonymous short reads need to be identified by sequence comparison to a set of reference sequences. This identification is required to analyze the microbial composition and biological diversity of the sample. We briefly introduce a new algorithm for evolutionary (phylogenetic) placement of short reads under the Maximum Likelihood criterion and implement it in RAxML. While this algorithm is significantly more accurate than plain pair-wise sequence comparison it can become highly compute-intensive when a typical number of 100,000 reads and more need to be placed into an existing phylogenetic tree. Therefore, we deploy multi-grain parallelism to improve parallel efficiency of this algorithm on 16-core and 32-core architectures. Via this multi-grain approach, we achieve parallel execution time improvements of 25% and super-linear speedups on 16 cores, as well as near-linear speedups and improvements exceeding 50% on 32-cores on two large real-world microbial datasets. Evolutionary placement of 100,000 reads into a tree with more than 4,000 taxa now only requires less than 2 hours of execution time on 32 cores.

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**ABSTRACT:**We present an evolutionary placement algorithm (EPA) and a Web server for the rapid assignment of sequence fragments (short reads) to edges of a given phylogenetic tree under the maximum-likelihood model. The accuracy of the algorithm is evaluated on several real-world data sets and compared with placement by pair-wise sequence comparison, using edit distances and BLAST. We introduce a slow and accurate as well as a fast and less accurate placement algorithm. For the slow algorithm, we develop additional heuristic techniques that yield almost the same run times as the fast version with only a small loss of accuracy. When those additional heuristics are employed, the run time of the more accurate algorithm is comparable with that of a simple BLAST search for data sets with a high number of short query sequences. Moreover, the accuracy of the EPA is significantly higher, in particular when the sample of taxa in the reference topology is sparse or inadequate. Our algorithm, which has been integrated into RAxML, therefore provides an equally fast but more accurate alternative to BLAST for tree-based inference of the evolutionary origin and composition of short sequence reads. We are also actively developing a Web server that offers a freely available service for computing read placements on trees using the EPA.Systematic Biology 03/2011; 60(3):291-302. · 12.17 Impact Factor - SourceAvailable from: uni-muenchen.de[Show abstract] [Hide abstract]

**ABSTRACT:**Plant beneficial microorganisms, such as arbuscular mycorrhiza fungi (AMF), increasingly attract scientific and agronomic attention due to their capacity to increase nutrient accessibility for plants and to reduce inorganic fertilizer requirements. AMF are thought to form symbioses with most land plants, obtaining carbon from the autotrophic host whilst enhancing uptake of poorly available nutrients. The species of AMF are mainly identified by spore morphology, which is time consuming, requires expertise and is rarely applicable to AMF identification in roots. Molecular tools such as analysis of standardized DNA fragment sequences may allow the recognition of species through a ‘DNA barcode’, which may partly overcome this problem. The focus of this study was to evaluate different regions of widely used rDNA repeats for their use as DNA barcodes for AMF including the small subunit rRNA gene (SSU), the internal transcribed spacer (ITS) and the large subunit rRNA gene (LSU). Closely related species in the genus Ambispora, members of which have dimorphic spores, could not be separated by analysis of the SSU region, but of the ITS region. Consequently, the SSU was not used for subsequent analysis, but a DNA fragment covering a small part of the SSU, the entire ITS region and about 800 bp of the LSU (SSUmCf-LSUmBr fragment) was analysed, providing phylogenetic resolution to species. New AMF specific primers for these potential barcoding regions were developed and can be applied, without amplification of non-target organisms, for AMF species determination, including identification from field and root samples. Analyses based on the application of the SSUmCf-LSUmBr fragment showed that the widely used AMF model organism Glomus sp. DAOM197198 (formerly called Glomus intraradices) is not conspecific with Gl. intraradices. The SSUmCf-LSUmBr fragment clearly provides a much higher species resolution capacity when compared with the formerly preferred ITS and LSU regions. Further study of several groups of AMF species using different regions of the SSUmCf-LSUmBr fragment revealed that only the complete SSUmCf-LSUmBr fragment allowed separation of all analysed species. Based on these results, an extended DNA barcode covering the ITS region and parts of the LSU region is suggested as a DNA barcode for AMF. The complete SSUmCf-LSUmBr fragment sequences can serve as a database backbone for also using smaller rDNA fragments as barcodes. Although the smallest fragment (approximately 400 bp) analysed in this study was not able to discriminate among AMF species completely, such short regions covering the ITS2 or LSU D2 regions, respectively, would most likely be suitable for community analyses with 454 GS-FLX Titanium sequencing, providing that the analyses is based on the longer DNA sequences.01/2010; - [Show abstract] [Hide abstract]

**ABSTRACT:**Selective sweep detection localizes targets of recent and strong positive selection by analyzing single nucleotide polymorphisms (SNPs) in intra-species multiple sequence alignments. Substantial advances in wet-lab sequencing technologies currently allow for generating unprecedented amounts of molecular data. The increasing number of sequences and number of SNPs in such large multiple sequence alignments cause prohibiting long execution times for population genetics data analyses that rely on selective sweep theory. To alleviate this problem, we have recently implemented fine- and coarse-grain parallel versions of our open-source tool OmegaPlus for selective sweep detection that is based on the ω statistic. A performance issue with the coarse-grain parallelization is that individual coarse-grain tasks exhibit significant run-time differences, and hence cause load imbalance. Here, we introduce a significantly improved multi-grain parallelization scheme which outperforms both the fine-grain as well as the coarse-grain versions of OmegaPlus with respect to parallel efficiency. The multi-grain approach exploits both coarse-grain and fine-grain operations by using available threads/cores that have completed their coarse-grain tasks to accelerate the slowest task by means of fine-grain parallelism. A performance assessment on real-world and simulated datasets showed that the multi-grain version is up to 39% and 64.4% faster than the coarse-grain and the fine-grain versions, respectively, when the same number of threads is used.Algorithms and Architectures for Parallel Processing. 01/2012;

Page 1

Evolutionary Placement of Short Sequence Reads

on Multi-Core Architectures

Alexandros Stamatakis, Zsolt Komornik, Simon A. Berger

Dept. of Computer Science, Technische Universit¨ at M¨ unchen, Boltzmannstr. 3, 85748 Garching b. M¨ unchen, Germany

Email: stamatak@in.tum.de, z.komornik@gmail.com, simon.berger@in.tum.de

Abstract—The application of high performance computing

methods in bioinformatics becomes increasingly important be-

cause of the masses of data generated by novel short-read DNA

sequencers. One important application of such short reads, is

the analysis of microbial communities where the anonymous

short reads need to be identified by sequence comparison to

a set of reference sequences. This identification is required to

analyze the microbial composition and biological diversity of the

sample. We briefly introduce a new algorithm for evolutionary

(phylogenetic) placement of short reads under the Maximum

Likelihood criterion and implement it in RAxML. While this

algorithm is significantly more accurate than plain pair-wise

sequence comparison it can become highly compute-intensive

when a typical number of 100,000 reads and more need to be

placed into an existing phylogenetic tree. Therefore, we deploy

multi-grain parallelism to improve parallel efficiency of this

algorithm on 16-core and 32-core architectures. Via this multi-

grain approach, we achieve parallel execution time improvements

of 25% and super-linear speedups on 16 cores, as well as near-

linear speedups and improvements exceeding 50% on 32-cores on

two large real-world microbial datasets. Evolutionary placement

of 100,000 reads into a tree with more than 4,000 taxa now only

requires less than 2 hours of execution time on 32 cores.

Index Terms—Evolutionary Placements; Short Reads; Maxi-

mum Likelihood; Pthreads; Multi-Grain Parallelism; RAxML;

I. INTRODUCTION

The application of high performance computing methods to

bioinformatics applications faces two challenges: the many-

core revolution and the biological data flood that is driven by

novel wet-lab sequencing techniques. These new sequencing

techniques can generate more than 100,000 short read DNA

sequences with a length ranging between 30 to 450 nucleotides

in a single run. Besides full-genome assembly (see, e.g.,

[1]), another important application is the in-vivo sampling of

microbial communities, e.g., in the human gut [2] or on human

hands [3]. Given the short reads, the first step in the analysis

process of microbial communities consists of identifying the

anonymous reads by determining to which of the known

(identified) organism sequences they are most closely related

to. This is usually done by pair-wise sequence comparisons,

i.e., each of the short reads is compared against all reference

sequences via a respective BLAST [4] search or, e.g., pairwise

sequence alignment [5]. An assignment of anonymous reads to

known organisms allows for comparison of microbial commu-

nities from different samples [2], for instance, samples from

different habitats, and allows to determine their respective

phylogenetic diversity [6].

Here we present the parallelization of a novel phyloge-

netic identification (placement) algorithm for anonymous reads

(henceforth denoted as Query Sequences (QS)), using a set

of full length Reference Sequences (RS). While, as already

mentioned, the most straight-forward approach for identifying

the QS consists of using similarity methods such as BLAST

and pair-wise alignment, this standard approach faces some

limitations: Firstly, the QS might be too short to yield sufficient

signal for a correct placement. Secondly, this approach can

yield misleading assignments of QS to RS if the RS sample

does not contain sequences that are sufficiently closely related

to the QS. Thus, the most important argument against a

BLAST-based approach is, that it will not unravel potential

problems in the sampling of the RS (see [7]). For instance,

given two RS A and B, a QS Q may be assigned to A by

BLAST, while it is in reality most closely related to a RS

C that was not included in the reference sequence set. Thus,

many studies of microbial communities deploy phylogenetic

(evolutionary) trees for QS placement, despite the significantly

higher computational cost. If a QS falls into an inner branch

of the phylogenetic reference tree comprising the RS, i.e., it is

not located near a leave of the tree that represents a currently

living species, this indicates that the sampling of the RS is

insufficient to correctly place and capture the diversity of the

QS. This also provides vital information about the parts of the

reference tree whose sampling needs to be improved. In fact,

this information can be used to guide efforts for full-length

DNA sequencing in order to improve the taxon sampling of the

organisms under study and fill in the missing parts in our view

of evolutionary history. Note that, the accuracy of phylogeny

reconstruction increases with taxon sampling [8].

To date, phylogeny-based placement of reads (environmen-

tal samples) is conducted as follows: the QS are aligned with

respect to a Reference Alignment (RA) for the Reference

Sequences (RS), and then inserted into the Reference Tree

(RT), either via a complete de novo tree reconstruction, a

constrained tree search (using the RT as a constraint topology),

or some fast and approximate QS addition algorithm as

implemented for instance in ARB [9]. This approach yields

a fully resolved bifurcating tree that can comprise more than

10,000 sequences from a single gene, typically 16S or 18S

rRNA [2], [3]. The reconstruction of large single-gene trees is

hard because of a weak signal, i.e., reconstruction accuracy

decreases for trees with many short sequences [10], [11],

and even more so for short-read QS. Hence, we advocate a

Page 2

different approach that only computes the optimal insertion

branch, i.e., the optimal placement, for every QS in the RT

with respect to its Maximum Likelihood (ML [12]) score. This

approach is faster than a de novo tree reconstruction and at

the same time significantly more accurate than BLAST, in

particular when the taxon sampling of the reference tree is

sparse or inadequate with respect to the sample. Moreover,

using appropriate additional heuristics, the placement algo-

rithm is as fast as BLAST. The complexity of both alternative

approaches is O(qr), where q is the number of QS and r

the number of RS. Results regarding the accuracy assessment

of our novel algorithm including a detailed comparison to

BLAST, impact of QS length on accuracy etc. are outside

the scope of this paper. While we briefly sketch the results

here, a draft manuscript (currently under review) providing

a detailed experimental setup, accuracy assessment, and run-

time comparison is available also available [13].

The evolutionary placement algorithm, including the par-

allelization described here, is already available in the latest

open source code release of RAxML (Randomized Axelerated

Maximum Likelihood [14], version 7.2.6, February 2010,

http://wwwkramer.in.tum.de/exelixis/software.html) which is a

widely used program for phylogenetic inference (over 6,700

downloads from distinct IPs to date, approximately 700 cita-

tions of the three main papers according to Google Scholar).

In this paper we focus on deploying multi-grain parallelism

on multi-core architectures to accommodate the computational

requirements of the placement algorithm by using three real-

world 16S rRNA datasets with 4,412 [3], 16,307 [2], and

100,627 QS (unpublished) that are classified into a single-

gene bacterial reference tree with 4,874 species and a length

of 1,287 base pairs. We show that fine-grain parallelism in

the phylogenetic likelihood function [15], [16] does not scale

well for the typical input datasets (many taxa, few sites)

of our algorithm and propose a multi-grain approach that

dynamically switches from a fine-grain parallelization scheme

to a more coarse-grain scheme during program execution.

This multi-grain approach yields near-linear and even super-

linear speedups as well as parallel run time improvements of

more than 50% on two general purpose multi-core systems.

Using our evolutionary placement method in combination with

multi-grain parallelism, it is possible to conduct phylogenetic

classification runs on large numbers of QS (100,627) in under

2 hours on a 32 core system.

The remainder of this paper is organized as follows: In

Section II we cover related work on parallel phylogenetic

inference. In Sections III and IV we describe the ML method

and provide an abstract description of the novel evolutionary

placement algorithm. In Section V we summarize the results

of the accuracy assessment for the sequential algorithm and

the compare it to plain sequence similarity-based approaches.

Thereafter, we outline the multi-grain parallelization strategy

(Section VI). The experimental setup and results are covered

in Section VII and we conclude in Section VIII.

II. RELATED WORK

This paper introduces a novel algorithm for phylogenetic

placement. Thus, we are not aware of any directly related work

that covers the problems associated with the parallelization of

such an algorithm.

Own previous work mainly focused on exploiting loop-

level parallelism for the ML function (see next Section) in

RAxML on shared memory and multi-core systems [16], the

IBM Cell [17], and the IBM BlueGene/L [18]. While the fine-

grain loop-level parallelism of the ML function scales well on

very large (in terms of number of base pairs) phylogenomic

alignments up to thousands [18] of processors, scalability

is limited for large single-gene alignments. Minh et al [19]

implemented a hybrid OpenMP/MPI version of IQPNNI that

exploits loop-level and coarse-grain parallelism. The ML func-

tion in GARLI [20] has also been parallelized with OpenMP.

III. THE MAXIMUM LIKELIHOOD MODEL

The input of a phylogenetic analysis consists of a multi-

ple sequence alignment with n sequences (taxa/tips) and m

alignment columns (also called alignment sites). The output

is an unrooted binary tree; the n taxa are located at the

leaves of the tree and the inner nodes represent common

(extinct) ancestors. The branch lengths represent the relative

time of evolution between nodes in the tree. In order to

compute the likelihood on a fixed tree topology, one requires

several Maximum Likelihood (ML) model parameters: the

instantaneous nucleotide substitution matrix Q which contains

the transition probabilities for time dt between nucleotide (4x4

matrix) characters and the prior probabilities of observing

the nucleotides: πA,πC,πG,πT. Finally, one also requires the

2n − 3 branch lengths in the unrooted tree topology.

Given these parameters, to calculate the likelihood on a

fixed tree, one needs to compute the entries for all ancestral

probability vectors at inner nodes, that contain the probabilities

P(A),P(C),P(G),P(T), of observing an A, C, G or T

at a site c, where c = 1...m, of the ancestral probability

profile of this inner node. The probability vectors are computed

bottom-up from the tips towards a virtual root that can be

placed into any branch of the tree via the Felsenstein pruning

algorithm [12]. If the substitution model is time-reversible,

the likelihood score will be the same for any placement of

the virtual root and thereby greatly simplifies the computation

of the likelihood score. However, there have been proposals,

for relatively easy to compute non-reversible substitution mod-

els [21].

Given a parent (ancestral) node k, and two child nodes

i and j (with respect to the virtual root), their probability

vectors⃗L(i)and⃗L(j), the respective branch lengths leading to

the children biand bj, and the transition probability matrices

P(bi),P(bj), the probability of observing an A at position c of

the ancestral (parent) vector⃗L(k)

A(c) is computed as follows:

⃗L(k)

A(c) =(

T

∑

S=A

PAS(bi)⃗L(i)

S(c))(

T

∑

S=A

PAS(bj)⃗L(j)

S(c))

(1)

Page 3

The transition probability matrix P(b) for a given branch

length is obtained from Q via P(b) = eQb, i.e., via an Eigen-

value/Eigenvector decomposition. Once the two probability

vectors⃗L(i)and⃗L(j)to the left and right of the virtual root

(vr) have been computed, the likelihood score l(c) for an

alignment column c can be calculated as follows, given the

branch length bvrbetween nodes i and j:

l(c) =

T

∑

R=A

(πR⃗L(i)

R(c)

T

∑

S=A

PRS(bvr)⃗L(j)

S(c))

(2)

The overall score is then computed by summing over the

per-column log likelihood scores:

LnL =

m

∑

c=1

log(l(c))

(3)

An important property of the likelihood function is, that sites

evolve independently, i.e., all entries c of a vector⃗L can be

computed simultaneously, which is the main source of fine-

grain parallelism [22], [23].

In order to compute the Maximum Likelihood value on

a fixed tree all individual branch lengths and the Q matrix

must also be optimized via ML. For Q the most common

approach in state-of-the-art ML implementations consists of

using Brent’s algorithm [24]. To evaluate changes in Q the

entire tree needs to be re-traversed, i.e., a full tree traversal

needs to be conduct to re-compute the likelihood. For the

optimization of branch lengths the Newton-Raphson method is

commonly used (see, e.g., [25]). The branches of the tree are

repeatedly visited and optimized one by one until the induced

branch length change is smaller than a pre-defined ϵ. The

Newton-Raphson method only operates on a single pair of

likelihood vectors⃗L(i),⃗L(j)that are located at either end of

the branch to be optimized. Note that in the parallel version of

RAxML, a reduction operation to compute the overall score

(over all columns) for the first and second derivative is required

at every iteration of the Newton-Raphson procedure. Hence,

we need to synchronize threads between all iterations of the

Newton-Raphson method for optimizing a single branch in a

fine-grain parallelization of the likelihood function [26].

An important issue is the assignment of memory space

for ancestral probability vectors. There exist two alternative

approaches: a separate vector can be assigned to each of the

three outgoing branches of an inner node, or only one vector

can be assigned to each inner node. In the latter case, the

probability vectors always maintain a rooted view of the tree,

i.e., they are oriented towards the current virtual root of the

tree. In the case that the virtual root is then relocated to a

different branch (for instance to optimize the respective branch

length), a certain number of vectors, for which the orientation

to the virtual root has changed need to be re-computed. If

the tree is traversed in an intelligent way for branch length

optimization, the number of probability vectors that will need

to be re-computed can be kept to a minimum. An example

for this data organization as used in RAxML is provided in

Figure 1.

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Virtual Root

Virtual Root

re−locate virtual root

re−compute

re−locate and

Fig. 1.Rooted organization of the probability vectors at inner nodes.

Virtual Root

Thread1Thread0

Sum0

Sum1

w

x

y

z

z

y

x

w

Probability Vector

AAG−TT

AGG−−T

AGGGTT

GGG−−T

AGG−−G

AAGG−T

Reduction = Sum0 + Sum1

Sync

Fig. 2.

tree with given branch lengths.

Parallel fine-grain computation of the likelihood score on a given

The main bulk of all of the above likelihood computations

consists of for-loops over the length m of the multiple

sequence input alignment which typically require 95% of total

execution time in all state-of-the-art likelihood-based software

packages. The general parallelization scheme of the Pthreads-

based version of RAxML is outlined in Figure 2 for a full

tree traversal. We use a cyclic distribution of the m alignment

columns to allow for better load-balance in phylogenomic

datasets that can contain DNA as well as AA (protein); protein

data requires more FLOPS per alignment site.

The master thread steers the tree search and orchestrates

the optimization of the branch lengths and model parameters.

For the model parameter optimization phase, which is relevant

to the algorithm presented here, the tree needs to be fully

traversed to optimize Q. Hence, the master thread generates a

full tree traversal list, that remains fixed during the model pa-

rameter optimization process because the tree topology is not

being changed. When a model parameter has been changed,

every worker thread can independently update its fraction of

the vector entries for the full tree traversal and the threads

only need to be synchronized when the virtual root is reached

(see Figure 2). Therefore, every thread can conduct a relatively

large fraction of independent work per alignment column dur-

ing the model parameter optimization phase. Since the branch

length optimization process requires several iterations at every

individual branch, the synchronization to computation ratio for

Page 4

branch length optimization is less favorable (see [26]). While

this fine-grain parallelization approach is highly efficient and

scales up to 1,024 CPUs for large phylogenomic analyses [18],

[27] with m ≫ 1,000, scalability for single-gene analyses

with m ≈ 1,000 is limited. In Section VI we provide solutions

to this single-gene scalability problem for the evolutionary

placement algorithm. Note however, that similar solutions

using a scatter-gather operation on probability vectors can

potentially also be applied to improve parallel efficiency and

scalability for standard single-gene ML searches.

IV. EVOLUTIONARY PLACEMENT ALGORITHM

As already mentioned the input for our algorithm consists

of a reference tree comprising r full-length RS (Reference

Sequences), and a comprehensive alignment that contains the

r RS as well as the q QS (Query Sequences). The QS can,

e.g., be aligned to the reference alignment with ARB [9] or

NAST [28]. The sequence alignment programs MAFFT [29]

and MUSCLE [30] can also be deployed to extend and merge

alignments. One key assumption is that the reference tree is

biologically well-established or has been obtained via a pre-

ceding thorough ML analysis of the RS. Initially, the algorithm

will read in the reference tree and reference alignment and

mark all sequences of the alignment that are not contained in

the reference tree as QS. Thereafter, the ML model parameters

and branch lengths on the reference tree will be optimized.

Once the model parameters and branch lengths have been

optimized, the placement algorithm is invoked. It will visit

the 2r − 3 branches of the reference tree in depth first-

order, starting at an arbitrary branch of the tree. At each

branch, initially the probability vectors of the reference tree

to the left and the right will be computed (if they are not

already oriented towards the current branch). Thereafter, the

program will successively insert (and remove again) one QS

at a time into the current branch and compute the likelihood

(we henceforth denote this as insertion score) of the respective

tree containing r + 1 taxa. The insertion score is stored in a

q × (2r − 3) table that keeps track of the insertion scores for

all q QS into all 2r−3 branches of the reference tree. Hence,

the complexity of our placement algorithm is O(rqm) since

we need to compute q × (2r − 3) insertion scores. Note that,

the time for the computation of an insertion score is a linear

function of the alignment length m (see Equations 2 and 3).

In order to more rapidly compute the per-branch insertions

of the QS we use an approximation that is comparable to the

Lazy Subtree Rearrangement (LSR) moves that are deployed

in the standard RAxML search algorithm [14]. After inserting

a QS into a branch of the reference tree we would normally

need to re-optimize all branch lengths of the thereby obtained

new—extended by one QS—tree topology to obtain the Maxi-

mum Likelihood insertion score. Instead, we only optimize the

three branches adjacent to the insertion node of the QS (see

Figure 3) before computing the insertion score. In analogy

to the LSR moves, we also use two methods to re-estimate

the three branches adjacent to the insertion branch, a fast

method that does not make use of the Newton-Raphson method

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b1r b2r

bq

Query Sequence

Reference tree

Branch lengths

to be changed

Fig. 3.

Sequences (QS) into the reference tree.

Local Optimization of branch lengths for the insertion of a Query

reference sequencequery sequence

QS0

QS1

QS2

RS0

RS1 RS3

RS2

reference tree

Best−scoring

Assignment

of Query Sequence

Fig. 4.

using a 4-taxon reference tree.

Evolutionary placement of 3 Query Sequences (QS0, QS1, QS2)

and a slow method. The fast insertion method just splits the

branch of the reference tree brinto two parts br1and br2by

setting br1 :=√br, br2 :=√br, and the branch leading to

the QS is set to bq:= 0.9, where 0.9 is the default RAxML

value to initialize branch lengths. The slow method repeatedly

applies the Newton-Raphson procedure to all three branches

br1,br2,bq until the branch length changes are ≤ ϵ, where

ϵ = 0.00001.

The output of this procedure is the input reference tree,

enhanced by assignments of the QS to the respective best-

scoring insertion branch. This means that QS are attached to

branches that yield the best insertion score for the respective

QS. Hence, the algorithm will return a potentially multi-

furcating tree, if more than one QS is assigned to the same

branch. An example output tree for 4 RS and 3 QS is provided

in Figure 4.

Moreover, RAxML can also conduct a standard phyloge-

netic bootstrap [31], i.e., repeat the evolutionary placement

procedure several times under slight perturbations of the

input alignment. This allows to account for uncertainty in the

placement of the QS as shown in Figure 5. Thus, a QS might

be placed into different branches of the reference tree with

various levels of support. For the Bootstrap replicates we also

use additional heuristics to accelerate the insertion process.

During the insertions on the original input alignment we keep

track of the insertion scores for all QS into all branches of

the reference tree. For every QS we can then sort the insertion

Page 5

0.2

0.8 1.0

0.7

0.3

BS support value

RS0

RS1

RS2

RS3

QS0

QS1

QS2

QS2

QS0

Fig. 5.

a 4-taxon reference tree with Bootstrap support.

Evolutionary placement of 3 query sequences (QS0, QS1, QS2) into

branches by their scores and only conduct insertions for a

specific QS into the 10% best-scoring branches. Evidently,

this reduces the number of insertion scores to be computed

per QS by 90% and yields an approximately ten-fold speedup.

The bootstrap procedure can for instance be used to compute

the distribution of the phylogenetic diversity of the QS in the

environmental sample.

Finally, we are currently also experimenting with methods

(that are already implemented) to simultaneously align and

insert QS to a specific branch of the reference tree using a

Maximum Likelihood and Maximum Parsimony-based [32]

dynamic programming algorithms. This will allow for direct

and more convenient analyses of environmental reads, because

users will be able to conduct the alignment and placement

step in one single run. Evidently, the complexity will increase

to O(rqm2) because of the dynamic programming approach.

Therefore, the framework we develop here can be used to

integrate and accelerate the future release of the simultaneous

alignment and placement procedures.

V. PERFORMANCE OF SEQUENTIAL ALGORITHM

As already mentioned, a detailed performance analysis as

well as a description of the experimental setup to assess the

accuracy of our algorithm is available [13]. We assessed the

accuracy of our evolutionary placement algorithm on 8 real-

world (mostly single-gene) datasets that were hand-aligned by

biologists. Initially, we computed best-known ML trees and

bootstrap support values on these alignments using RAxML.

We then pruned a single taxon (a virtual Query Sequence) at

a time from these trees. Thereafter, we tried to re-insert the

virtual Query Sequence into the tree using our evolutionary

placement algorithm, BLAST, and a simple sequence com-

parison algorithm. This sequence-based comparison algorithm

makes use of the aligned sequences to compute all pair-wise

edit distances to the taxa of the Reference Alignment that are

located at the leaves of the tree. We only selected taxa (virtual

QS) for pruning that had bootstrap support ≥ 75% ([13] for

selection details and rationale), i.e., taxa with a sufficiently

well-supported position in the tree. In addition, we artificially

shortened the virtual QS in order to generate sequences that

20406080100

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

% non−gap characters

average node−dist

simple seq. based

EPA

Fig. 6.

short-read sequences as a function of the percentage of non-gap characters,

averaged over all test datasets.

Accuracy of sequence similarity-based and ML-based placement of

have the same length as 454 reads, i.e., approximately 200-400

base pairs.

The accuracy was then assessed by determining the number

of nodes that lie between the original “true” pruning position

of the virtual QS and the respective placement branch as com-

puted by our algorithm, BLAST and the sequence comparison

algorithm. In Figure 6, we depict the average node distance

from the correct insertion position, averaged over all datasets.

The x-axis indicates the percentage of non gap-characters

in the virtual—artificially shortened—Query Sequences with

respect to the full alignment length. Figure 6, clearly shows

that our algorithm is twice as accurate as the sequence-based

placement approach.

In a second set of experiments we shortened the full-

length virtual query sequence by generating artificial paired-

end reads, i.e., just kept 100 base-pairs of nucleotide data at

the left and right end of the full length sequence and inserted

gaps in the middle. We then used our placement algorithm

and BLAST to compute insertion positions for those query

sequences. In Figure 7, we provide a histogram for a DNA

dataset of 855 taxa that depicts the number of inserted query

sequences at distances of 0, 1, 2, etc. nodes away from the

original “correct” position. This histogram shows that ML-

based placement is also significantly more accurate than a

BLAST-based approach.

Finally, we have developed several additional heuristics to

accelerate the evolutionary placement algorithm (similar to

the procedure for the acceleration of bootstrapping outlined

in Section IV), that are only 1.5-2 times slower than BLAST

(see Figures 10.a and 10.b in [13]), but are almost as accurate

as the slow standard evolutionary placement algorithm. The

parallelization of all these additional heuristics and the boot-

strapping procedure is analogous to the general parallelization

scheme which we present in the following Section.

Page 6

0 1 3 4 6 7 9 11 131618212326 283133 3638

EPA

BLAST

node−distance from correct insertion position

count

0

50

100

150

200

Fig. 7.

sequences as a function of the percentage of non-gap characters, averaged

over all test datasets.

Accuracy of BLAST- and ML-based placement of short-read

VI. MULTI-GRAIN PARALLELIZATION

The current release of RAxML (v7.2.6) contains a full fine-

grain Pthreads-based parallelization of the likelihood function

(see Section III and [16]). The initial parallelization of the

evolutionary placement algorithm was hence straight-forward

because we used the existing parallel framework. However,

the scalability of the fine-grain parallelization is limited on

datasets with few sites such as the real-world single gene

bacterial datasets we use in our experiments.

The main parallelization challenge lies in the two different

phases of the program that exhibit varying degrees of paral-

lelism: In the initial phase during which ML model parameters

are optimized, the branch length optimization procedure is

hard to be further parallelized because of intrinsic dependen-

cies between individual branch length optimization steps. The

insertion phase consists of computing the q×(2r−3) insertion

scores of the QS on the reference tree and is easier to paral-

lelize. Hence we need to deploy multi-grain parallelism using

the fine-grain parallelization scheme for the initial phase and

a more coarse-grain parallelization strategy for the insertion

phase. One can parallelize the insertion phase by essentially

conducting all q × (2r − 3) insertion score computations

simultaneously. Here, we parallelize by branches, i.e., we use a

cyclic distribution of reference tree branches to threads where

every thread computes insertion scores for insertions of all q

QS into one specific branch. Given the number of branches

in real-world reference trees (9,745 insertion branches, see

Section VII) which is expected to further increase in the future,

this represents a sufficiently fine-grain source of parallelism for

this algorithm. In order to be able to compute QS insertions

simultaneously we require to compute and store all pairs of

probability vectors for all branches in the tree. Hence, for

every branch we need a contiguous (remember that for the

fine-grain parallelization we use a cyclic distribution of the

probability vector columns) probability vectors attached to the

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contigoous vector

contigoous vector

contigoous vector

contigoous vector

Gather

Gather

Gather

Gather

contigoous vector

contigoous vector

re−locate virtual root

Virtual Root

Virtual Root

re−locate &

re−compute

Fig. 8.

the reference tree.

Multi-grain gather operation of the per-branch probability vectors on

left and right end of each branch. This allows a single thread

to independently compute all insertions of the QS into one

branch, since only those two vectors are required to compute

the insertion score.

In order to store and compute contiguous probability vectors

we initially allocate a data-structure of length 2r − 3 that

corresponds to the total number of branches in the reference

tree. For every entry of this branch data structure we allocate

two contiguous (full-length) probability vectors that are used

to store the probability vectors to the left and the right of that

branch in the reference tree. In order to fill the per-branch

probability vectors we proceed as follows: We traverse the

entire reference tree in depth-first order and place the virtual

root into the branch that is currently being visited and re-

compute the probability vectors (if necessary) to the left and

right of the current branch. Once the strided vectors using

the cyclic probability vector column distribution have been

computed, we conduct a gather operation to store the per-

thread columns contiguously in the vectors attached to the

branch data structure. This traverse and gather procedure is

outlined in Figure 8. The overhead of this full tree traversal

and the gather operation is negligible (less than 0.5% of overall

execution time) compared to the overall execution time as well

as the time required for initial model parameter optimization

in fine-grain mode. Once the probability vector pairs for all

branches have been stored, we can simply distribute work to

threads in a cyclic way, i.e., thread 0 will insert all QS into

branch 0, thread 1 will insert all QS into branch 1, etc.

An additional rationale for deploying a per-branch par-

allelization strategy is a planned distributed memory paral-

lelization of the evolutionary placement algorithm with MPI

(Message Passing Interface). If we parallelized by insertion

sequences we would need to hold all insertion branch vectors

in the memory of every process which might lead to memory

shortage on systems such as the IBM BlueGene/L or Blue-

Gene/P.

VII. EXPERIMENTAL SETUP & RESULTS

We used three real-world 16S rRNA datasets with 4,412 [3]

(HAND dataset), 16,307 [2] (GUT dataset), and 100,627

Page 7

(unpublished) QS that are classified into a single-gene bacterial

reference tree with 4,874 species and a reference alignment

length of 1,287 base pairs. Those datasets represent typical

current use cases for our algorithm and the two smaller ones

are freely available for download together with the multi-

grain parallelization of the source-code at http://wwwkramer.

in.tum.de/exelixis/software.html. As test systems for assessing

scalability we used a 4-way quad-core AMD Barcelona system

with a total of 16 cores running at 2.2 GHz and 128 GB

of main memory, and a Sun x4600 8-way quad-core AMD

Shanghai system with a total of 32 cores running at 2.7 GHz

and 64 GB of main memory.

Computational experiments were conducted as follows: On

the AMD Barcelona system we executed one sequential run

and Parallel Fine-Grain (PFG) as well as Parallel Multi-Grain

(PMG) runs on 2, 4, 8, and 16 cores for the fast insertion

method. On the x4600 we executed the same runs, but up to

32 cores. We also executed two runs using the slow insertion

method with 16 threads for the PFG and PMG parallelization

on the 16-core system. The execution time for the slow method

using the PFG approach on the HAND dataset was 35 hours

compared to only 22 hours using the PMG implementation.

Thus, for the slower (and more accurate) insertion algorithm

on the HAND dataset we achieve a parallel efficiency im-

provement exceeding 35%. The execution times for the slow

method on the larger GUT dataset where 139 hours (PFG) and

76 hours (PMG) respectively, which corresponds to a parallel

run time improvement of 45%.

In Figure 9 we depict the speedups for the fast insertion

algorithm using the PFG and PMG parallelizations on the

Barcelona system for datasets HAND and GUT. Figure 9

shows that the PFG version scales reasonably well on the

16-core system and even achieves super-linear speedups due

to improved cache efficiency up to 8 threads. However, as

outlined in Sections III and VI the scalability of the fine-

grain approach is limited for single-gene alignments. The

impact of an increasing amount of synchronization events per

computation in the PFG parallelization is demonstrated by

the sub-linear speedups on 16 cores. The increase in parallel

efficiency of the PMG over the PFG approach on 16 cores for

the fast insertion method is slightly lower than for the slow

insertion method, but we still achieve a 25% improvement on

16 cores. This is because the contribution of the less scalable

fine-grain initial model optimization phase to overall execution

time is larger for the fast than for the slow insertion method.

Nonetheless, speedups are significantly super-linear up to 16

cores for the PMG version. The overall speedups are slightly

lower on the 32-core system (Figure 10), because of the less

scalable fine-grain initial model optimization phase; stand-

alone placement without model optimization scales linearly.

However, the increase in parallel efficiency of the PMG over

the PFG approach with 32 cores exceeds 50%.

Figures 9 and 10 show that the parallel efficiency of the evo-

lutionary placement algorithm is generally higher on the AMD

Barcelona system. The reason for this is that the improved

cache efficiency of the parallel algorithm has a larger impact

0

2

4

6

8

10

12

14

16

18

20

22

0 2 4 6 8 10 12 14 16

Speedup

# Threads

linear speedup

GUT PMG

HAND PMG

GUT PFG

HAND PFG

Fig. 9.

system using the fast insertion algorithm.

Speedups for overall execution times on a 16-core AMD Barcelona

0

5

10

15

20

25

30

35

0 5 10 15

# Threads

20 25 30 35

Speedup

linear speedup

GUT PMG

HAND PMG

GUT PFG

HAND PFG

Fig. 10.

using the fast insertion algorithm.

Speedups for overall execution times on a 32-core x4600 system

on the Barcelona cores that have 2MB L3 caches compared

to 6MB L3 caches on the Shanghai cores. In Figure 11 we

plot the speedup of the Shanghai over the Barcelona CPUs as

a function of the number of threads. When only one Shanghai

core is used, the program is almost twice as fast as on a

Barcelona core which can not be explained by the higher clock

rate and improved micro-architecture alone. The main cause is

the three times larger L3 cache size. If the number of threads is

increased and cache misses are thereby decreased, the speedup

converges to 1.23 which corresponds to the clock rate ratio

(2.7GHz/2.2GHz).

Finally, we also conducted experiments on a challenging

dataset with 100,627 QS to explore the limits of our approach

on the x4600 system. We obtained overall speedups of 14.5

and 27.3 for fast insertion runs with 16 and 32 cores respec-

tively. On 32 cores the overall runtime for the placement of

100,627 QS is less than 1.5 hours which makes our approach

a viable and more accurate alternative to BLAST and other

sequence comparison-based approaches that do not take into

account the evolutionary history of the sequences under study.

VIII. CONCLUSION & FUTURE WORK

We have introduced a new, accurate, algorithm for ML-

based evolutionary placement of short reads which is already

being used by several research groups (personal communi-

Page 8

1

1.2

1.4

1.6

1.8

2

0 2 4 6 8 10 12 14 16

Speedup

# Threads

overall speedup

insertion speedup

clock frequency ratio

Fig. 11.

architecture.

Speedup of the 32-core (Shanghai) over the 16-core (Barcelona)

cation) for real-world studies of microbial communities. We

show that fine-grain parallelism does not scale well for the

analysis of real-world microbial datasets and propose a more

appropriate multi-grain parallelization approach. Our paral-

lelization yields super-linear speedups on multi-core systems

with small L3 caches and parallel run time improvements of

25% up to 50% compared to the fine-grain approach.

Current work covers the full integration of the multi-grain

parallelization with RAxML, i.e., the support of all data types

(morphological, DNA, secondary structure, protein data) and

models of rate heterogeneity. We also plan to devise an

MPI-based parallelization. Future work, will focus on using

multi-grain parallelism for an insertion algorithm that can

simultaneously place and align the QS.

ACKNOWLEDGMENT

We wish to thank R. Knight and M. Hamady for providing

test datasets, M. Stark and C.v. Mehring for productive dis-

cussions on algorithm design, and B. Moret for access to the

Barcelona system.

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