Mason – A Read Simulator for Second

Generation Sequencing Data

Manuel Holtgrewe

B-10-06
October 2010

FACHBEREICH MATHEMATIK UND INFORMATIK

SERIE B • INFORMATIK
 Abstract
We present a read simulator software for Illumina, 454 and Sanger reads.
Its features include position specific error rates and base quality values. For
Illumina reads, we give a comprehensive analysis with empirical data for the
error and quality model. For the other technologies, we use models from the
literature. It has been written with performance in mind and can sample
reads from large genomes. The C++ source code is extensible, and freely
available under the GPL/LGPL.

1. Introduction
Second generation sequencing technologies yield DNA sequence data at unprecedented
high throughput. The generated data has many applications, including genome re-
sequencing, and structural variant detection.
Various sequencing technologies are commercially available: Instruments based on py-
rosequencing by Roche/454 Life Sciences [MEA+ 05], reversible terminator chemistry by
Illumina [BBS+ 08], and sequencing by oligonucleotide ligation and detection by Applied
Biosystems are examples of second-generation sequencing. Helicos Biosciences offers the
first commercial instrument for single-molecule sequencing.
The Short Read Archive (SRA) offers huge numbers of freely available read data.
However, when developing, testing and evaluation software that processes sequencing
data, using real-world data only is not desirable. While evaluating performance on
real-world data is indispensable, simulated data nicely complements real data.
First, the original sample location in the genome is not known for real-world data
while it is available for simulated data. Second, the data in the SRA is often more than
one year old since authors publish their data in the SRA after the publication of their
paper. This means that the technology used for reads in the SRA is older, e.g. with
shorter read numbers org having no mate pairs. Third, simulation allows to consider
certain characteristics of data in an isolated way. For example, one can increase the
error rate in the simulated reads to show the robustness of an algorithm.
Because of this, many authors use simulated data for evaluating their algorithms.
Many authors use their own, possibly ad-hoc, software for generating such reads. How-
ever, there are also some publications that only deal with the simulation of reads.
In [Mye99], Myers describes celsim, a program for the simulation of Sanger reads.
Since the genomes of important model organisms were not known at that time, it also
allows the synthesis of genomes. In [ROA+ 08], Richter et al. describe MetaSim, a
program for the simulation of reads. The focus of MetaSim is metagenomics and it
allows to sample reads from a larger set of genomes and also to artificially let these
genomes evolve. Recently, in [BML+ 10] , Balzer et al. describe flowsim, a simulator for
pyrosequencing reads, based on the analysis of empirical data.
Table 1 shows properties of these read simulators and Mason, the software described
in this paper. The source code of Mason is freely available, it supports the generation

1
 Feature celsim MetaSim flowsim Mason
Source code available X X
Sanger reads X X X
Sanger qualities X X
454 reads X X X
454 qualities X X
Illumina reads X X
Illumina qualities X

Table 1: Properties of read simulation software. Citations are: celsim [Mye99], MetaSim
[ROA+ 08], flowsim [BML+ 10]. Mason is described in this article.

of Illumina, 454 and Sanger reads. Our group has successfully used the simulator for
benchmarking software for read mapping, read correction and transcript quantification.
The read simulator has been implemented using C++ using the SeqAn library.

2. Simulation Models
The simulation program framework is described in Section 3. In this section, we describe
the simulation model parts that depend on the simulated technology: The length of the
physical sample (reference sequence infix), the model for simulating sequencing errors
and base quality values.
Following George Box’ words “All models are wrong but some are useful,” the aim
is not to find a model that fits reality completely. Rather, we want to find simple that
show important characteristics of the simulated sequencing technologies.

2.1. Model for Illumina Reads

Illumina sequencing works by synthesizing one strand of the reads base by base: The
reads are arranged in beads of identical reads. Chemicals flow along the beads in cycles
(A, T, C, G). In each cycle, one base binds in each read. The binding of the bases emits
light which is recorded by a camera. Based on the recorded points of light emission, it
is decided which base has been bound most probably.
In our model, we ignore the death of beads and assume each bead emits a signal in
each step. However, the steps can be faulty: The signal can be misinterpreted, not be
detected, or the signal can be emitted in a later step. The faults correspond to single
base polymorphisms, deleted, and inserted bases in the read.
It was shown in [DLBH08] that there are context dependent biases for errors. We
ignore this fact for our model, as is usually the case in read simulation. However, we
incorporate the positon of a base in the read, for the simulation of errors: Because of
properties of the chemical process, the probability for an error raises with every step.
Our first draft model (which we will revise below) is thus:

2
 Read Set Species Length [bp] Submission Platform
SRR026674 fly 36 2009/09/28 Illumina GA 2
SRR026675 fly 36 2009/09/28 Illumina GA 2
SRR026676 fly 36 2009/09/28 Illumina GA 2
SRR049254 fly 100 2010/05/20 Illumina GA 2
SRR038098 yeast 20 2010/03/16 Illumina GA 2
SRR003673 yeast 36 2008/08/12 ”Illumina” (1G?)

Table 2: Properties of the read sets chosen for determining position-depending probabil-
ities.

• The read length ` is constant.

• The probability for mismatches, insertions, deletions at position i in the read is

given by pm (i), pd (i), and pi (i).

• On mismatches, the replacing base is chosen at random, excluding the replaced

base.

We performed an experimental study to determine these position specific probabilities.

We use the program RazerS [WER+ 09] for mapping reads with full sensitivity against
the reference genome with a minimal identity of 90 %. RazerS thus finds all best hits
(see [HWRE10] for a definition of “all best hits”) of up to 10 % errors (relative to the
read length) for each read. This means that for each read, all matches with the lowest
distance are found. The resulting multi read alignment is then analyzed for positional
mismatches, inserts and deletions.
When speaking about matching, mismatching, inserted and deleted bases, we are
always considering the multi read alignment returned by RazerS. We are aware that
this is biased towards the alignment algorithms and scoring schemes used inside RazerS.
However, any semiglobal alignment schema will have a bias.
We ran the analysis on Illumina reads of fly and yeast.
We chose flybase release 5.27 of Drosophila melanogaster as the reference sequence.
As reads, we chose the read sets with SRA accession numbers SRR049254 (100 bp reads)
and SRR026674-SRR026676 (36 bp reads). We concatenated chromosomes NC 001133-
NC 001148 and NC 001224 for Saccharomyces cerevisiae as the reference sequence. As
reads, we chose SRR038098 (20 bp reads) and SRR003673 (36 bp reads). This is sum-
marized in Table 2.

2.1.1. Overall Errors

First, consider Table 3. This table shows the percentage of reads with a given number
of errors for different read sets (the numbers for SRR026674-6 are very similar). As we
can see, for the 100 bp reads, about 25 % of all reads have more than 2 errors.

3
 errors percentage
SRR038098 SRR003673 SRR026674 SRR049254
0 95.39 78.41 74.77 43.52
1 3.58 11.98 17.25 20.85
2 1.02 5.61 5.64 10.87
3 0.01 3.97 2.33 6.61
4 0 0.01 0.01 4.51
5 0 0 0 3.34
6 0 0 0 2.68
7 0 0 0 2.23
8 0 0 0 1.94
9 0 0 0 1.73
10 0 0 0 1.55
11 0 0 0 0.05

Table 3: Percentage of reads with a given number of errors.

2.1.2. Positional Errors

We show each of the following plots in two versions.

• One for read sets SRR026674, SRR049254, SRR038098, and SRR003673 (sets A)
to show the variance between the results from different studies.

• One for the read sets SRR026674, SRR026675, and SRR026676 (sets B) to show
the variance within the same experiment.

The plots themselves can be found in Appendix A. Figures 1, 2, and 3 show the
positional mismatch, insertion and deletion error rates. We can see there are differences
between the insertion error rates in sets A (Figure 1a) and even those from the same
experiment (sets B, Figure 1b).
The reads from sets B were sequenced in 2008, being one of the first reads to be
sequenced in paired-end mode outside Illumina ([Tho10]). The authors consider these
reads to be of low quality mostly due to source prep. Newer but not yet published read
sets have a much better quality, according to the authors. Note that while the con-
sistently growing error rates in the order SRR026674, SRR026675, SRR026676 suggest
instrument drift, the authors did not confirm this. The runs were not necessarily done
in this order. Two runs were made at the same day, one on the next. A new flow cell is
used for each run, so cleaning is not an issue either.
Positional insertion rates can be seen in Figure 2. The rates being 0 for the first and
last base are caused by the alignment algorithm in RazerS: At the end of the program,
the reads are aligned semiglobally against the reference sequence and the gap penalties
are slightly larger than mismatch penalties. This means, alignments like these:

4
 ... CAACAAC-AACAACAACAA-CAACAACAACAA ...
|||||||||||
AAACAACAACAAA

are replaced with alignment like these:

... CAACAACAACAACAACAACAACAACAACAA ...

|||||||||||
AAACAACAACAAA

The half-moon shape of the error rates in Figure 2b for sets B also have to be explained
to be alignment algorithm artifacts. Note that the insert rates are one order of magnitude
lower than the mismatch rates. The closer a mismatch occurs towards the ending of an
alignment, the higher is the probability that an inserted base shifts the leading or trailing
bases to match to a non-significant but random match. In sets SRR049254, SRR038098,
and SRR003673, the right tip of the half-moon is much larger than the left one. This
can be explained by the fact that the insertion rates are much higher towards the end
of the read, and for these reads, the mismatch error rate grows strong towards the end
than for read sets B.
A similar explanation holds for the shapes of the deletion rate curves shown in Figure 3.
We hope to correct such alignment algorithm biases by a multi read realignment program
such as seqcons [RKD+ 09] in the future. Currently, bugs in this program prevent us from
using it in a whole-genome setting.
We consider the insert and deletion probabilities to be independent of the position
and calibrate them with average rates from the middle of the analyzed reads. Thus, our
error model for Illumina reads is:

• The read length ` is constant.

• The probability for mismatches at position i in the read is given by pm (i).
• The probability for insertions and deletions is the same at each position and given
by pi and pd .
• On mismatches, the replacing base is chosen at random, excluding the replaced
base.

The position dependent mismatch probability can be given explicitely as an empirical

curve. Another possibility is to specify it as a two-piece affine function in the position
relative to the read length. From 0 up to a position x with a flat slope and from x to 1
with a steeper slope. The empirical data suggests 2/3 as the value for x.

2.1.3. Positional Qualities

Figures 4, 5, 6, 7, 8 show the positional mean qualities of matching, mismatching,
inserted bases and those of the bases before/after deleted bases. The vertical bars show
the standard deviations.

5
 We first note that the qualities before and after deleted bases hardly differ. Second, the
qualities for inserted bases roughly follow those of neighbours of deleted bases. Third,
the qualities of matching and mismatching bases don’t differ much at the beginning but
quickly separatetowards the ending. The qualities of inserted and deleted bases lie in
between, first following the quality of matching bases up the relative position of 1/3.
In Section 2.1.2, we described methodological problems of determining whether a base
is inserted/deleted or mismatching. Because of this, inserted and deleted bases could in
fact be mismatching or matching bases. For now, we decide only to differentiate between
mismatches and the rest of error types. Hopefully, a realignment step can clarify whether
this is adequate or not.
We decide to model the qualities as position specific normal distributions. One for
mismatching bases, and one for all other bases. The position depending means follow a
linearly falling ramps, the standard deviations follow a linearly raising ramp.

2.2. Model for 454 LS Reads

454 pyrosequencing works by synthesizing one strand of a read homopolymer by ho-
mopolymer (where a homopolymer is the repetition of the same base). For each syn-
thesized hompolymer, light is emitted with a light intensity depending on its length `.
Background noise is simulated as light with log-normally distributed intensity.
In the original publication [MEA+ 05], the authors give normal distributions with a
mean of ` and standard deviation of k · ` where k is a fixed proportionality factor. The
read simulator MetaSim, described in [ROA+ 08] models the standard deviation as by
k · ` by default, to be more consistent with “basic statistics.” We do the same. We model
background noise as a log-normal distribution with mean 0.2 and standard deviation 0.1.
We remind the reader that often, the formula for the log-normal distribution function
is given with the mean and standard deviation of the underlying normal distribution.
In this case, mean and standard deviation of the log-normal distribution have to be
transformed.
In the simulation, the light intensities are simulated following the normal distributions,
with an additive background noise. Consistent with MetaSim and flowsim [BML+ 10],
we call bases within one flow cycle using a simple Bayes base caller. We use the log-odds
transformed error probability as returned by the Bayes base caller as quality values, as
does [BML+ 10].

2.3. Model for Sanger Reads

For Sanger reads, we follow the model from celsim as proposed in [Mye99]:

• Read lengths are either uniformly sampled from an interval or normally distributed
with given mean and standard deviation.

• Insertions, deletions and mismatches are randomly distributed with position de-
pendent probabilities. The position dependent probabilities are computed by ramp
functions with configureable probabilities at the beginning and the end.

6
 • The quality simulation is the same as for Illumina reads, as described in Sec-
tion 2.1.3.

3. Simulation Framework
This section gives a short overview of the implementation. The read simulation frame-
work works as follows:
First, the reference sequence is loaded. Alternatively, a random sequence with a given
background distribution can be generated.
Second, model specific parameters are computed or loaded from a file. For example,
the empirical error distribution for Illumina reads can be loaded in this step.
Third, haplotypes are simulated from the reference sequence: The reference sequence
is taken and changes are applied to it (actually, we only store a list with modifications for
shorter running times). At each position, a base substition, an insertion, or a deletion is
applied with user defined probabilites. The length of insertions and deletions is randomly
picked, inserted bases are picked uniformly at random.
Fourth, the reads are simulated. Section 2 describes the parts that depend on the
simulated technology. For each read:

• Pick the read length, depending on the simulation model.

• Pick physical sample location and haplotype.

• Depending on the simulation model, generate edit string and a buffer with inserted,
substituted bases.

• Simulate qualities, depending on the edit string and the simulation model.

• If mate pairs are to be simulated then pick location for the mate and perform the
upper steps for the mate.

• Add metainformation about the reads sample location, originally sample reference
infix and edit string into the sequence descriptor.

Fifth, the reads are written out into a FASTA/FASTQ file. Optionally, the program
writes out the alignment of the reads against the reference sequence in a SAM [LHW+ 09]
file.

4. Conclusion and Future Work

4.1. Conclusion
We have presented a read read simulator for Illumina, 454 and Sanger reads. It incorpo-
rates position-specific error rates and base quality distributions. Its source code is freely
available hat http://www.seqan.de/projects/mason.html.

7
4.2. Future Work
Using empirical distributions and simulating degradation for 454 reads following [BML+ 10]
would be very useful. Furthermore, a future version should support the simulation of
SOLiD color space reads.
The simulation of Helicos reads is another point. However, direct access to raw
sequencing Helicos sequencing data would be necessary for this, similar to the work
in [BML+ 10].

5. Acknowledgements
I thiank Anne-Kathrin Emde, David Weese, and Knut Reinert for enlightening discus-
sions on 2GS technologies.

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A. Positional Error Rate Plots

6
SRR026674_1
SRR049254_1
SRR038098_1
SRR003673
5

4
error rate [%]

0
0 20 40 60 80 100
position

(a) Positional mismatch error rates in read sets A.

2.2
SRR026674_1
SRR026675_1
2 SRR026676_1

1.8

1.6
error rate [%]

1.4

1.2

0.8

0.6

0.4
0 5 10 15 20 25 30 35
position

(b) Positional mismatch error rates in read sets B.

Figure 1: Positional mismatch error rates.

11
 0.7
SRR026674_1
SRR049254_1
SRR038098_1
0.6 SRR003673

0.5
error rate [%]

0.4

0.3

0.2

0.1

0
0 20 40 60 80 100
position

(a) Positional insert error rates in read sets A.

0.07
SRR026674_1
SRR026675_1
SRR026676_1
0.06

0.05
error rate [%]

0.04

0.03

0.02

0.01

0
0 5 10 15 20 25 30 35
position

(b) Positional insert error rates in read sets B.

Figure 2: Positional insert error rates.

12
 0.2
SRR026674_1
SRR049254_1
0.18 SRR038098_1
SRR003673
0.16

0.14

0.12
error rate [%]

0.1

0.08

0.06

0.04

0.02

0
0 20 40 60 80 100
position

(a) Positional delete error rates in read sets A.

0.045
SRR026674_1
SRR026675_1
0.04 SRR026676_1

0.035

0.03
error rate [%]

0.025

0.02

0.015

0.01

0.005

0
0 5 10 15 20 25 30 35
position

(b) Positional delete error rates in read sets B.

Figure 3: Positional delete error rates.

13
B. Positional Quality Value Plots

60
SRR026674_1
SRR049254_1
SRR038098_1
SRR003673
50

40
mean quality

30

20

10

0
0 20 40 60 80 100
position

(a) Positional match quality values in read sets A.

60
SRR026674_1
SRR026675_1
SRR026676_1
50

40
mean quality

30

20

10

0
0 5 10 15 20 25 30 35
position

(b) Positional match quality values in read sets B.

Figure 4: Positional match base quality values.

14
 60
SRR026674_1
SRR049254_1
SRR038098_1
SRR003673
50

40
mean quality

30

20

10

0
0 20 40 60 80 100
position

(a) Positional mismatch quality values in read sets A.

60
SRR026674_1
SRR026675_1
SRR026676_1
50

40
mean quality

30

20

10

0
0 5 10 15 20 25 30 35
position

(b) Positional mismatch quality values in read sets B.

Figure 5: Positional mismatch base quality values.

15
 60
SRR026674_1
SRR049254_1
SRR038098_1
SRR003673
50

40
mean quality

30

20

10

0
0 20 40 60 80 100
position

(a) Positional insert quality values in read sets A.

60
SRR026674_1
SRR026675_1
SRR026676_1
50

40
mean quality

30

20

10

0
0 5 10 15 20 25 30 35
position

(b) Positional insert quality values in read sets B.

Figure 6: Positional inserted base quality values.

16
 60
SRR026674_1
SRR049254_1
SRR038098_1
SRR003673
50

40
mean quality

30

20

10

0
0 20 40 60 80 100
position

(a) Quality values of bases before deleted ones in read sets A.

60
SRR026674_1
SRR026675_1
SRR026676_1
50

40
mean quality

30

20

10

0
0 5 10 15 20 25 30 35
position

(b) Quality values of bases before deleted ones in read sets B.

Figure 7: Quality values of bases before deleted ones.

17
 60
SRR026674_1
SRR049254_1
SRR038098_1
SRR003673
50

40
mean quality

30

20

10

0
0 20 40 60 80 100
position

(a) Quality values of bases after deleted ones in read sets A.

60
SRR026674_1
SRR026675_1
SRR026676_1
50

40
mean quality

30

20

10

0
0 5 10 15 20 25 30 35
position

(b) Quality values of bases after deleted ones in read sets B.

Figure 8: Quality values of bases after deleted ones.

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