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CALIFORNIA STATE LTNIVERSITY SAN MARCOS
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OF THE REQUIREMENTS FOR THE DEGREE
MASTER OF SCIENCE
IN
COMPUTER
PROJECT TITLE:
AUTHOR:
SCIENCE
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Prediction of Biomineralization Proteins Using Machine Learning Models
Chad Davies
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511212015
THE PROJECT HAS BEEN ACCEPTED BY THE PROJECT COMMITTEE IN
PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF
SCIENCE TN COMPUTER SCIENCE.
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PROJECT COMMITTEE CHAIR
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DATE
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Prediction of Biomineralization Proteins Using
Machine Learning Models
Chad Davies
Spring 2015
1
CS 698 – Master’s Project in Computer Science
California State University San Marcos (CSUSM)
Spring 2015
Master’s Project Committee Members:
Dr. Xiaoyu Zhang, CSUSM Department of Computer Science
Dr. Ahmad Hadaegh, CSUSM Department of Computer Science
2
Table of Contents
Prediction of Biomineralization Proteins Using Machine Learning Models .................................. 1
Abstract ....................................................................................................................................... 5
1
Introduction ...................................................................................................................... 6
2
Background – Related Work ............................................................................................ 7
3
Data and Methods............................................................................................................. 9
3.1
Program Architecture ................................................................................................ 9
3.2
Data Preparation...................................................................................................... 10
3.3
Feature Computations ............................................................................................. 14
3.4
Normalization ......................................................................................................... 16
3.5
Machine Learning Models ...................................................................................... 17
3.5.1
4
Fivefold Cross Validation................................................................................ 18
Results ........................................................................................................................... 18
4.1
Statistical Measures ................................................................................................ 18
4.2
Analysis Five-fold Cross Validation ....................................................................... 20
4.2.1
RBF-SVM Fivefold Run Results For Best Parameters ................................... 20
4.2.2
L-SVM Fivefold Individual Run Results ........................................................ 20
4.2.3
K-NN Fivefold Run Results For Best Parameter ............................................ 21
4.2.4
Average Fivefold Run Results ......................................................................... 22
3
4.3
Results on E. huxleyi Proteins ................................................................................ 25
5
Conclusion and Future Work ........................................................................................ 28
6
Appendix ....................................................................................................................... 29
7
Appendix - Unknown Protein Sequence Results .......................................................... 32
8
References ..................................................................................................................... 35
4
Abstract
Biomineralization is used by Coccolithophores to produce shells made of calcium carbonate that
still allow photosynthetic activity to penetrate the shell because the shells are transparent. This
project tried to build machine learning models for predicting biomineralization proteins. A set of
biomineralization proteins and a set of random proteins were selected and processed using the
cd-hit program to cluster similar sequences. After the processing, 138 biomineralization proteins
and 1,000 random proteins were used for building the models. Three sets of features with an
overall count of 248 features were then computed and normalized to build prediction models by
using three different machine learning algorithms: Radial Basis Function Support Vector
Machine (RBF-SVM), Linear Support Vector Machine (L-SVM), and K-Nearest Neighbor (KNN). The models were trained and tested using Five-fold cross validation, which indicated a
MCC of 0.3834 for the K-NN, 0.3906 for the L-SVM, and the best result being 0.3992 for the
RBF-SVM. The accuracy of the K-NN and RBF-SVM models were comparable to that of
machine learning models developed for other types of proteins. The models were then applied to
predict biomineralization proteins in Emiliania huxleyi. The K-NN predicted 86 proteins, the
RBF-SVM predicted 3,441 proteins, and the L-SVM predict 11,671 proteins as possible
biomineralization proteins For the E. huxleyi dataset, with 59 shared among all three algorithms,
which suggests K-NN might be the best model in this study.
5
1
Introduction
Living organisms create minerals which harden or stiffen tissues by a process known as
Biomineralization. Biomineralization is a biological process common in many different species,
from single-cellular algae to large vertebrates. It was believed that Biomineralization proteins in
different species share some chemical, biological and structural features. In this project, I will be
working on building prediction models for the proteins that are responsible for the
Biomineralization process, and applying it to identify potential Biomineralization proteins in
Emiliania huxleyi, a prominent type of Coccolithophore.
A Coccolithophore is a small cell that is only a few micrometers across, and this cell is
encapsulated by small plates that form a shell called a coccolith. This Coccolithophores use the
Biomineralization process called coccolithogenesis to create coccoliths made of calcium carbonate
which are transparent and allow organisms to have a hardened shell that still allows for
photosynthetic activity.
A Biomineralization and a random dataset were used in the machine learning models. The
Biomineralization dataset was obtained from the AmiGO2 [2] resource, and the random dataset
was obtained from UniProt [6]. These datasets were fed into CD-HIT [5] where all the sequences
were clustered into similar sequences. Three sets of features, ProtParam, a delta function, and the
AAIndex, were selected from both datasets; then, the features were normalized between a range
of 0 and 1 & -1 and 1 before the machines were used to classify them.
Three machine learning models were used; two of them were Support Vector Machines
(SVM) with one being Radial Basis Function Support Vector Machine (RBF-SVM) and the
second being Linear Support Vector Machine (L-SVM). The third machine learning model used
6
was the K-Nearest Neighbor (K-NN). The best unit of measurement for comparing the three
models was Matthews correlation coefficient (MCC). The best result was the RBF-SVM with a
MCC of 0.3992 and the second best result was the L-SVM with a MCC of 0.3906 which are very
close. K-NN had an MCC of 0.3834 which is somewhat worse than the other two.
The report describes the works related to protein sequencing in the background section.
Data origination and transformation are also discussed in detail along with the architecture
developed for the three machine learning models implemented. Results are discussed and
compared to the other related works in this report. Future improvements are presented for others
to investigate.
2
Background – Related Work
The idea of using a SVM to identify Biomineralization proteins came from the paper
“BLProt: prediction of bioluminescent proteins based on support vector machine and relieff
feature selection” [3]. This paper used a SVM to predict Bioluminescent proteins and obtained
favorable results. Since the paper’s results were satisfactory using SVM, it made sense for us to
utilize the same approach to Biomineralization data to see if our results would mimic their
success.
In addition to using SVM from the paper above, the AAIndex feature vector was also
used for its similar implementation between Biolumination from their paper and
Biomineralization used by this project. This project utilizes the 544 physicochemical properties
in the AAIndex to predict favorable Biomineralization proteins. These 544 physicochemical
7
properties are fed into a feature vector along with 2 other feature vectors which are consumed by
the SVM algorithms to predict favorable Biomineralization proteins.
K-Nearest Neighbors (K-NN) was used to a great extent by “BLKnn: A K-Nearest
Neighbor Method For Predicting Bioluminescent Proteins” [4] to predict Bioluminescent
proteins. This second paper uses the K-NN algorithm to find proteins that are Bioluminescent
instead of the SVM used in the first paper. Also used from the second paper above are the
original 9 physicochemical properties: BULH740101, EISD840101, HOPT810101,
RADA880108, ZIMJ680104, MCMT640101, BHAR880101, CHOC750101, and COSI940101
which were used as a base feature list before inserting uncorrelated indexes. Then the AAIndex
list was utilized to make a new feature list containing the original 9 and all non-correlated
indexes. The delta function was also used from the above paper to obtain the delta features. This
used an offset to compare different positions in a sequence to see if there is a correlation. In this
project, the delta features were used in conjunction with the AAIndex features. Finally,
ProtParam, an existing Python package specifically designed to analyses protein sequences, was
used from the same paper to calculate the protein analysis. This project also applied: molecular
weight, aromaticity, instability index, flexibility, isoelectric point, and secondary structure
fraction as features. Therefore, this paper combines the data features and the machine learning
techniques from both of the papers in an attempt to more accurately predict Biomineralization
proteins.
8
3
Data and Methods
3.1
Program Architecture
Once the two data files random and Biomineralization are obtained, they are transformed
through the use of an existing program which clusters like records. The remaining data then goes
through a data preparation stage and a data normalization stage. Then, the data is fed into the
three machine learning algorithms, and the resulting output is used to compare each of the three
results. See Figure 1 below for an architectural diagram. Please see the Appendix for a general
guide of how to run the program and get these results.
9
High Level Architecture
Biomineralization
Data
Random Data
Transformation
- CD-HIT – Cluster Data
- Data Preparation
- Normalization
Machine Learning Models
L-SVM
RBF-SVM
K-NN
Results
&
Comparison
Figure 1. High Level Architecture
3.2
Data Preparation
The Biomineralization data, for this project, was sourced from the AmiGO 2 website [2]
using specifically the “biomineral tissue development” data [2]. This data was downloaded, and
then the column of identifiers in the data was used in UniProt [6] to obtain a FASTA format file
10
that contained the full 839 Biomineralization sequences. A FASTA formatted file is an industry
standard representation of the Biomineralization sequences in which nucleotides or amino acids
are represented by single letter codes in plain text. The random dataset, which contained 547,599
sequences, was used in this project was also downloaded from UniProt [6] using the reviewed
(Swiss-Prot) FASTA format file link.
Sequence clustering algorithms can be used to group biological sequences that are similar
in some way. In our case, homologous sequence proteins are the data source which are usually
grouped into families. CD-HIT attempts to create representative sequences for each cluster and
assigns each sequence processed to an existing cluster if it is a close enough match. If it is not a
close enough match, then it becomes a representative sequence for a new cluster. Both the
Biomineralization sequences and the random sequences were run through CD-HIT [5] with a
threshold similarity score of 40% and a CD-HIT user guide recommended word size of 2. For the
Biomineralization data, CD-HIT was able to compile results all in one run, and after CD-HIT
was run, there were 138 sequences left. CD-HIT had to be run on the random dataset 4 times in
total due to the dataset being too large. First, the random dataset was split into 3 different
sections, and CD-HIT was run separately on each section. Then, CD-HIT was run once more on
the random data after recombining the results of the previous 3 CD-Hit runs. In the end, there
were 82,849 sequences in the random dataset of which 1,000 were randomly selected for training
and testing.
Details for obtaining the raw data, pre-processing or clustering the data and randomizing
the data for both the random and Biomineralization datasets are presented in Figure 2 and Figure
3 below.
11
Biomineralization Data Selection/Pre-Processing
AmiGo 2
Website File Download
Pull Identifier
data from
spreadsheet
Query UniProt website with
identifiers
FASTA File
Biomineralization data
839 Sequences
CD-HIT
Biomineralization Data
Clustered
138 Sequences
Figure 2. Biomineralization Data Selection Pre-Processing
12
Random Data Selection/Pre-Processing
UniProt Website
FASTA File Download
Random Data
547,599 Sequences
Break into 3
equal sized files
Random
A
Random
B
Random
C
CD-HIT
CD-HIT
CD-HIT
Random
Cluster A
Random
Cluster B
Random
Cluster C
Combined CD-HIT
Random Clustered
82,849 Sequences
Randomly Pick
1000 Sequences
Random Clusters
1000 Sequences
Figure 3. Random Data Selection Pre-Processing
13
3.3
Feature Computations
The next step was to use the bmfeat.py program that I had written in python to get three
sets of features. The program used the package ProtParam to parse the FASTA files that were
outputted from CD-HIT [5] into separate sequences, and then, it allowed us to parse the
sequences themselves into individual features. ProtParam also allowed us to parse the Amino
Acid Index (AAIndex) for easy use when obtaining the AAIndex features. The following feature
process occurs for both Biomineralization and the random dataset. In the data if a sequence had
an X, U, B, Z, or O, then it was skipped/removed from the program’s resulting data due to it
effecting the results/breaking the ProtParam feature package.
The first set of features are also from the ProtParam package which allowed us to get the
molecular weight, aromaticity, instability index, flexibility, isoelectric point, and secondary
structure fraction. For flexibility, 3 features were used: average, maximum and minimum. For
the secondary structure fraction, all of the three returned data values were used.
The following formula is the delta function: “Suppose a protein X with a sequence of L
amino acid residues: R1R2R3R4. . .RL, where R1 represents the amino acid at sequence position
1, R2 the amino acid at position 2, and so on. The first set, delta-function set, consists of λ
sequence-ordercorrelated factors, which are given by
14
Equation
Reference
𝐿𝐿−𝑖𝑖
1
𝛿𝛿𝑖𝑖 =
� ∆𝑗𝑗,𝑗𝑗+𝑖𝑖
𝐿𝐿 − 𝑖𝑖
(1)
𝑗𝑗=1
where i=1,2,3…λ, λ<L, and Δ j,j+i =Δ(Rj,Rj+i)=1 if Rj = Rj+1, 0 otherwise. We name these
features as {δ1, δ2,…δλ }.” [4]
The second set of features was for the delta function which has 27 features. The delta
features work using an offset from 1 to 27; it then compares the current value in the current
sequence against the value at x offset to the right of it in the sequence. This then occurs for the
entire sequence string, and if the current value matches the offset value then the counter is
incremented by 1. At the end, the counter is then divided by the total number of values in the
sequence; the result of this division is then stored in a feature list for later use.
The following formula is for the AAindex: “For each of 9 AAindex indices, we obtained
µ sequence-order-correlated factors by
Equation
Reference
𝐿𝐿−𝑖𝑖
1
ℎ𝑖𝑖 =
� 𝐻𝐻𝑗𝑗,𝑗𝑗+𝑖𝑖
𝐿𝐿 − 𝑖𝑖
(2)
𝑗𝑗=1
where i=1,2,3… µ, µ <L, and Hi,j =H(Ri)⋅ H(Rj). In this study, H(Ri) and H(Rj) are the
normalized AAindex values of residues Ri and Rj respectively” [4]
15
The third and final set of features is found using the AAIndex. First, the bmfeat.py
program goes through and finds all the default indexes from BULH740101, EISD840101,
HOPT810101, RADA880108, ZIMJ680104, MCMT640101, BHAR880101, CHOC750101, and
COSI940101 given by the BLKnn [4] and puts them in a feature list. It then goes through the
AAIndex file again adding every index to the feature list while ensuring there are no correlated
or duplicate indexes added to the feature list. Then the AAIndex file is opened a last time along
with each particular sequence. The feature list is then used to form features for every sequence.
The letter values in the sequence are mapped to the values in the AAIndex file. These mapped
values are all added up and divided by the total length of the sequence. This occurs for every
index in the feature list creating one feature every time. With all three feature lists completed for
each sequence, they will now be combined and outputted into a file ready for normalization.
3.4
Normalization
Before any machine algorithms consume the data, all the data was normalized. The
normalization process uses the maximum and minimum of the current feature in order to ensure
that all the data in a feature is represented fairly. This is accomplished by scaling all values in the
feature between -1 and 1 or 0 and 1. To normalize the data, both the random and
Biomineralization data were brought into the norm.py python program at the same time. This
was done in order to ensure that both datasets were normalized to the same degree utilizing the
same maximum and minimum on both datasets, so they are scaled the same. The total maximum
and minimum was then found and added onto both of the datasets. Then both datasets were
scaled to fit the total minimum and maximum. Then the total maximum and minimum were
removed from the dataset so it doesn’t alter the data. This was the process used on all the
16
ungrouped features. On the grouped features, the same process was used except the total
maximum and minimum is found for all the features in that set. For example the total maximum
and minimum would be found for all 27 of the delta features. Then, the total maximum and
minimum are applied to all 27 delta features at the same time.
3.5
Machine Learning Models
Two different machines were used to learn from the data. The first one was a SVM and it
has two parts: RBF-SVM and L-SVM. A Support Vector Machine is a supervised learning
model that attempts to classify the two classes (Biomineralization or Random data) passed in by
identifying patterns. A set of training data is placed on a multi-dimensional graph based on its
features. Then, the best possible pattern is identified and is used during testing to try and separate
the data into the two classes passed in. A separate test dataset is passed into the SVM and plotted
on the same graph, so depending on which side of the identifying pattern that each piece of data
is located; it will try to correctly classify the data. For the RBF-SVM, the parameter Gamma was
used to define the impact that the training data has while parameter c is used to define how
accurately it classifies the training data. The weight parameter was set to ‘auto’, and both the
Gamma and C parameters were scaled to find the optimal result. The L-SVM used the default
settings provided by SKLearn except for the weights parameter where ‘auto’ was used.
The third machine was the K-Nearest Neighbor algorithm which is also a supervised
learning method, and it classifies by examining its closest neighbors and how they have been
classified during the training phase. Each neighbor within a small neighborhood votes to classify
17
the current data under scrutiny, so that it can be defined as to which class (if any) it belongs to.
The K-NN algorithm that was used had ‘distance’ for its weights parameter and ‘auto’ for its
algorithm parameter, and the ‘neighborhood size’ parameter was tested on the data with a range
of 1 to 80.
3.5.1 Fivefold Cross Validation
Fivefold Cross Validation was used on both the random and Biomineralization datasets in
the training and testing phase. The data was split five ways by starting at location x = 0, 1, 2, 3,
and 4; then, every x + 5 locations was added to that particular set of data. These sets of data were
then used five different times with one of them being used for testing while the other four were
used for training.
4
Results
Before reviewing the results, it is best to explain the statistical measures: specificity,
sensitivity, accuracy, and MCC. Afterwards, I’ll review the results for each of the three
machines.
4.1
1.
Statistical Measures
Specificity: used in the program to correctly remove a condition.
Reference
Equation
𝒔𝒔𝒔𝒔𝒔𝒔𝒔𝒔𝒔𝒔𝒔𝒔𝒔𝒔𝒔𝒔𝒔𝒔𝒔𝒔𝒔𝒔 =
2.
𝑻𝑻𝑻𝑻
𝑻𝑻𝑻𝑻 + 𝑻𝑻𝑻𝑻
(3)
Sensitivity: used in the program to correctly identify a condition.
18
Reference
Equation
𝑺𝑺𝑺𝑺𝑺𝑺𝑺𝑺𝑺𝑺𝑺𝑺𝑺𝑺𝑺𝑺𝑺𝑺𝑺𝑺𝑺𝑺 =
3.
𝑻𝑻𝑻𝑻
𝑻𝑻𝑻𝑻 + 𝑭𝑭𝑭𝑭
(4)
Accuracy: calculated by looking at the correct 0 and 1 for the answer and comparing it to
the number produced by the program. If a 0 or 1 didn’t match up, then it isn’t counted but if it
does match up then it is added up and divided by the total number of comparisons.
Reference
Equation
𝑨𝑨𝑨𝑨𝑨𝑨𝑨𝑨𝑨𝑨𝑨𝑨𝑨𝑨𝑨𝑨 =
4.
𝑻𝑻𝑵𝑵 + 𝑻𝑻𝑻𝑻
𝑻𝑻𝑻𝑻𝑻𝑻
(5)
MCC: Matthews correlation coefficient takes into account the true/false positives and
negatives on two-class classifications with a returned range of -1 to 1 with negative one being the
complete opposite of what was predicted and positive one being exactly what was predicted.
Reference
Equation
𝑴𝑴𝑴𝑴𝑴𝑴 =
𝑻𝑻𝑻𝑻 𝒙𝒙 𝑻𝑻𝑻𝑻 − 𝑭𝑭𝑭𝑭 𝒙𝒙 𝑭𝑭𝑭𝑭
�(𝑻𝑻𝑻𝑻 + 𝑭𝑭𝑭𝑭)(𝑻𝑻𝑻𝑻 + 𝑭𝑭𝑭𝑭)(𝑻𝑻𝑻𝑻 + 𝑭𝑭𝑭𝑭)(𝑻𝑻𝑻𝑻 + 𝑭𝑭𝑭𝑭)
Equation Definitions:
TP: True Positive
TN: True Negative
FP: False Positive
FN: False Negative
TNC: Total Number of Comparisons
19
(6)
4.2
Analysis Five-fold Cross Validation
Below are the details for the individual Fivefold run results for the SVM’s and the K-NN
followed by a table displaying the average for each machine.
4.2.1 RBF-SVM Fivefold Run Results For Best Parameters
Overall the RBF-SVM had the highest average MCC out of all three machines that were
used. It also had the highest single run with an MCC of 0.5168 for any run of any machine. The
parameters used for RBF-SVM: C = 1000 and Gamma = 0.001.
Table 1. RBF-SVM Fivefold Run Results For Best Parameters
Fivefold Runs
Specificity
Sensitivity
Accuracy
MCC
1
79.27%
77.78%
78.90%
0.5168
2
72.29%
71.43%
72.07%
0.3898
3
71.08%
71.43%
71.17%
0.3775
4
80.72%
60.71%
75.68%
0.3937
5
55.42%
81.48%
61.82%
0.3185
4.2.2 L-SVM Fivefold Individual Run Results
On the average, the L-SVM had the lowest MCC score of all three algorithms. It also has
the lowest single run MCC score of 0.3386 of any run on any machine. And, its highest MCC
was 0.4694 somewhat below the highest single runs of each of the other machines.
20
Table 2. L-SVM Fivefold Individual Run Results
Fivefold Runs
Specificity
Sensitivity
Accuracy
MCC
1
74.39%
70.37%
73.39%
0.4009
2
79.52%
71.43%
77.48%
0.4694
3
69.88%
71.43%
70.27%
0.3654
4
79.52%
60.71%
74.77%
0.3791
5
57.83%
81.48%
63.63%
0.3386
4.2.3 K-NN Fivefold Run Results For Best Parameter
The K-NN was a close runner up to RBF-SVM with an average MCC of 0.3834 which is
less than 0.02 below RBF-SVM. The highest single run of the K-NN had an MCC of 0.4833
which was also 0.03 away from the highest result of the RBF-SVM. To obtain these results, the
nearest neighbor parameter used for K-NN was 8.
Table 3. K-NN Fivefold Run Results For Best Parameter
Fivefold Runs
Specificity
Sensitivity
Accuracy
MCC
1
91.46%
48.15%
80.73%
0.4418
2
92.77%
50.00%
81.98%
0.4833
3
92.77%
35.71%
78.38%
0.3522
4
96.39%
35.71%
81.08%
0.4335
5
77.11%
44.44%
69.09%
0.2062
21
4.2.4 Average Fivefold Run Results
The average of the best of the Fivefold runs for the RBF-SVM, L-SVM, and K-NN are
listed in the following table. Note that RBF-SVM provides the best overall result with an MCC
of 0.3992.
Table 4. Average Fivefold Run Results
Methods
Specificity
Sensitivity
Accuracy
MCC
RBF-SVM
71.76%
74.71%
71.93%
0.3992
L-SVM
72.22%
71.08%
71.91%
0.3906
K-NN
90.10%
42.80%
78.25%
0.3834
Table 4 came from the Fivefold average output of the machines. svm.py (All percentages are rounded to the
second decimal place.)
The results for specificity, sensitivity, accuracy, and MCC are the average of a 5 fold
cross validation technique for each different method. The K-NN had a respectable result of
excluding random data with a specificity of 90.10%, but achieved 42.80% sensitivity. The
accuracy was 78.25% correct while the MCC was 0.3834. The RBF-SVM attained a specificity
of 71.76% and a sensitivity of 74.71%. It also had an accuracy of 71.93%, but it had a slightly
better MCC 0.3992. The L-SVM had the lowest specificity of 72.22% and a sensitivity of
71.08%. The accuracy was 71.91% and the MCC was 0.3906.
Figure 4, below, is a graph of the parameters used and the MCC results of the RBF-SVM.
The RBF-SVM is controlled through the use of two parameters with one being Gamma which
defines the influence of the training data, and the other being C which either smooth or roughen
22
the classifying decision surface. You can see there are three hotspots for obtaining the best MCC.
The first one occurred when the log10(C) was 0 and the log10(Gamma) was -1. The second area
occurred between log10(C) 2 and 4, and the log10(Gamma) was between -5 and -2. The third
area occurred between a log10(C) of 5 and 6; and, log10(Gamma) was -4 to -5. The best MCC
occurred when log10(Gamma) was 0.001 and log10(C) was 1000. With added data for training
or with a more diverse C or Gamma, a greater MCC could be obtained.
Figure 4. RBF-SVM Parameter Plot
The graph below, Figure 5, displays the average MCC per number of nearest neighbors
for all the K-NN runs. The number of nearest neighbors is controlled by a parameter called to the
23
K-NN method. As you can see, the best results are obtained when the number of neighbors is
between 3 and 29. As the number of neighbors increases beyond 30, the MCC drops
significantly. If more data was available for training, a higher number of neighbors may increase
the MCC.
K-NN Averages - Not Best
MCC VS Number of Neighbors
0.45
0.4
0.35
MCC
0.3
0.25
0.2
0.15
0.1
0.05
0
1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31 33 35 37 39 41 43 45 47 49 51 53 55 57 59 61 63 65 67 69 71 73 75 77 79
Number of Neighbors
MCC
Figure 5. K-NN Averages
Comparing the results to “BLKnn: A K-Nearest Neighbors Method For Predicting
Bioluminescent Proteins” [4], their best method was a BLKnn with a specificity of 95.5%,
sensitivity of 74.9%, accuracy of 85.2%, and MCC of 0.719 while our best method was RBFSVM which has a specificity of 71.76%, sensitivity of 74.71%, accuracy of 71.93%, and MCC of
0.3992. Our method did not have as good a result as BLKnn which may be attributed to not using
24
feature selection. Another contributing factor might be the amount of data available for known
Biomineralization sequences. Our results were based on 44% less known data than their BLKnn
method which could make their training set much larger and more robust.
When comparing the results to “BLProt: prediction of bioluminescent proteins based on
support vector machine and releiff feature selection” [3], their best method was a BLProt with a
specificity of 84.2%, sensitivity of 74.5%, accuracy of 80.1%, and MCC of 0.5904. If you
compare the BLProt result to our RBF-SVM result above, the MCC is somewhat less than ours,
specificity is 13% less, sensitivity almost the same, and accuracy is 8% less. As with the
comparison with BLKnn, BLProt also used feature selection which may have improved their
results. Also, the BLProt has a dataset that had over twice as many known Bioluminescent
sequences which once again would make their training set much larger and more robust.
4.3
Results on E. huxleyi Proteins
Possible Biomineralization proteins in Emiliania huxleyi (Eh) were also analyzed through
the machine learning techniques RBF-SVM, L-SVM, and K-NN. To train the machine learning
algorithms, both the random and Biomineralization datasets from above were used since the
correct result was known for both datasets, and it increased the known data for the large amount
of unknown data to be processed. The Eh dataset contained 39,125 proteins that shrank to 19,391
possible candidates after running through CD-HIT [5]. The results varied greatly depending on
which machine was used. The K-NN identified 86 proteins, the RBF-SVM identified 3,441
proteins, and the L-SVM identified 11,671 proteins as possible Biomineralization proteins. The
K-NN parameter number of neighbors was set to 8 which was used because it had the best results
per Figure 4 above. The RBF-SVM parameters C and Gamma were set to 1,000, and 0.001
respectively; these two values had given the best results in our known training and test datasets
25
above. More could be done to improve this result such as having 10 to 20 fold increase in known
training data.
26
As seen in Figure 6 below, the overlapping Biomineralization sequences between K-NN
and RBF-SVM amounted to 59. The K-NN and L-SVM also had 75 Biomineralization
sequences, and RBF-SVM and L-SVM had 3,434 overlapping sequences. 59 sequences also
overlapped between all three machine learning techniques namely: K-NN, RBF-SVM, and LSVM. The sequence ID’s for the 59 are in Appendix - Unknown Protein Sequence Results.
These should be good candidates since all three algorithms predict these sequences would
produce Biomineralization.
Figure 6. Venn Diagram with relation to K-NN, RBF-SVM, L-SVM
27
5
Conclusion and Future Work
In conclusion, I achieved my results after downloading the data from AmiGO 2 website. I
then applied UniProt and CD-HIT to the data. Next, I used ProtParam, Delta with offset, and the
AAIndex to create three feature lists and combine them. Finally, I used Fivefold cross validation
along with RBF-SVM, L-SVM, and K-NN to get the resulting specificity, sensitivity, accuracy,
and MCC.
Since MCC takes into account both the true/false positives and negatives that the other
three calculations: specificity, sensitivity, and accuracy fail to consider, it is the optimal method
to interpret the results. Thus, the results show RBF-SVM is the best option of the three with a
MCC of 0.3992, the L-SVM was second best with a MCC of 0.3906, and the third was K-NN
with a MCC of 0.3834.
For future work, a feature selection method such as ReliefF could be added to cut down
on the number of features that were used. This helps identify the most valuable features and
reduces noise from useless features while also ensuring that the machines are not over trained. At
first, you test the most relevant features, and from there, you slowly add greater amounts of less
relevant features until you’ve tested all the features at the end. This will show you if having less
features will give you better results. Also, comparing the achieved results to other machine
learning techniques may be beneficial in finding a higher MCC score.
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6
Appendix
1. Directory Structure: Python programs contained all in one folder with a subdirectory
named ‘data’ which holds all the data files, both input and output.
2. First, CD-HIT should be run on both your random and Biomineralization dataset in order
to group sequence proteins.
a. To run CD-HIT, you can use the command:
cd-hit -i data.fasta -o dataout -c 0.4 -n 2
‘i’ = being the input file
‘o’ = being the output file
‘c’ = being the threshold
‘n’ = being the word length
b. The dataout file will now only contain data with a threshold that is less than 40%.
c. If your dataset is too large for a single CD-HIT run, then the dataset can be split
into sections with CD-HIT being run on each section. And finally, the dataset
sections can be recombined with CD-HIT being run on the recombined dataset to
give a complete output which would give us allcombinedout.fasta.
d. The output for the Biomineralization dataset would be dataout.fasta.
3. The random dataset is then run through reducesize.py with the input file being the
complete random dataset from CD-HIT above:
29
python reducesize.py allcombinedout.fasta random1000.fasta
The output will be 1,000 randomly selected sequences from the larger random dataset
with output file name being random1000.fasta.
4. The next step is to run the Biomineralization dataset and the random dataset through
bmfeat.py as input with the Amino Acid Index (filename: aaindex1 [1]) which utilizes the
sequences in the dataset to create an output file that contains three different kinds of
features:
python bmfeat.py aaindex1 dataout.fasta output.csv
python bmfeat.py aaindex1 random1000.fasta random1000output.csv
a. The first set of features are molecular weight, aromaticity, instability index,
flexibility, isoelectric point, and secondary structure fraction from the ProtParam
package.
b. The second set of features is from the delta set using the offset sequence
correlation.
c. And the third set of features utilizes the Amino Acid Index.
d. The file bseqnames.csv is generated automatically which is used as an input by
svmUnknown.py below.
30
5. Normalization then occurs in the norm.py file with the Biomineralization dataset and
random dataset as the input for processing. The normalization process converts the
features between -1 and 1 or 0 and 1 depending on what is needed:
python norm.py random1000output.csv output.csv randomnormalized.csv bionormalized.csv
6. Both normalized datasets are input into svm.py which contains the three machines: KNN, RBF-SVM, and L-SVM. This program then prints out the results for each machine.
python svm.py randomnormalized.csv bionormalized.csv
7. Finally for the unknown data, svmUnknown.py will print out the biomineralization
proteins that all three algorithms agree on.
python svmUnknown.py randomnormalized.csv bionormalized.csv newnormalized.csv
bseqnames.csv
31
7
Appendix - Unknown Protein Sequence Results
Below are the 59 overlapping sequences that all three machines predict will have
Biomineralization:
jgi|Emihu1|460906|estExtDG_fgeneshEH_pg.C_10018
jgi|Emihu1|349148|fgenesh_newKGs_kg.1__107__2692675:2
jgi|Emihu1|194122|gm1.100422
jgi|Emihu1|194627|gm1.100927
jgi|Emihu1|196201|gm1.400323
jgi|Emihu1|461962|estExtDG_fgeneshEH_pg.C_90081
jgi|Emihu1|462005|estExtDG_fgeneshEH_pg.C_90190
jgi|Emihu1|97687|fgeneshEH_pg.11__49
jgi|Emihu1|420966|estExtDG_fgenesh_newKGs_pm.C_150050
jgi|Emihu1|434550|estExtDG_fgenesh_newKGs_kg.C_190166
jgi|Emihu1|353397|fgenesh_newKGs_kg.21__47__EST_ALL.fasta.Contig4482
jgi|Emihu1|100011|fgeneshEH_pg.21__188
jgi|Emihu1|207124|gm1.2800276
jgi|Emihu1|450888|estExtDG_Genemark1.C_340220
jgi|Emihu1|464347|estExtDG_fgeneshEH_pg.C_450144
jgi|Emihu1|103373|fgeneshEH_pg.48__121
jgi|Emihu1|103846|fgeneshEH_pg.53__96
jgi|Emihu1|212934|gm1.5500172
jgi|Emihu1|452396|estExtDG_Genemark1.C_610117
jgi|Emihu1|104868|fgeneshEH_pg.63__34
jgi|Emihu1|311732|fgenesh_newKGs_pm.66__14
jgi|Emihu1|216420|gm1.6900142
jgi|Emihu1|453001|estExtDG_Genemark1.C_720170
jgi|Emihu1|465577|estExtDG_fgeneshEH_pg.C_740042
jgi|Emihu1|55258|gw1.83.55.1
32
jgi|Emihu1|221716|gm1.9800116
jgi|Emihu1|439634|estExtDG_fgenesh_newKGs_kg.C_1040054
jgi|Emihu1|222692|gm1.10400138
jgi|Emihu1|454304|estExtDG_Genemark1.C_1070053
jgi|Emihu1|69888|e_gw1.108.74.1
jgi|Emihu1|440018|estExtDG_fgenesh_newKGs_kg.C_1150051
jgi|Emihu1|454648|estExtDG_Genemark1.C_1180056
jgi|Emihu1|109721|fgeneshEH_pg.123__82
jgi|Emihu1|110082|fgeneshEH_pg.130__9
jgi|Emihu1|226409|gm1.13200060
jgi|Emihu1|363248|fgenesh_newKGs_kg.143__66__2703503:1
jgi|Emihu1|441219|estExtDG_fgenesh_newKGs_kg.C_1470040
jgi|Emihu1|441290|estExtDG_fgenesh_newKGs_kg.C_1490039
jgi|Emihu1|229461|gm1.15500130
jgi|Emihu1|230713|gm1.16600046
jgi|Emihu1|456264|estExtDG_Genemark1.C_1740072
jgi|Emihu1|468509|estExtDG_fgeneshEH_pg.C_1930030
jgi|Emihu1|233951|gm1.19900015
jgi|Emihu1|314847|fgenesh_newKGs_pm.209__9
jgi|Emihu1|366272|fgenesh_newKGs_kg.211__37__EST_ALL.fasta.Contig3109
jgi|Emihu1|443034|estExtDG_fgenesh_newKGs_kg.C_2140015
jgi|Emihu1|236083|gm1.23000015
jgi|Emihu1|237243|gm1.24700017
jgi|Emihu1|367484|fgenesh_newKGs_kg.261__3__2696944:1
jgi|Emihu1|239574|gm1.29100028
jgi|Emihu1|241880|gm1.34500018
jgi|Emihu1|469948|estExtDG_fgeneshEH_pg.C_3490015
jgi|Emihu1|445010|estExtDG_fgenesh_newKGs_kg.C_3850001
jgi|Emihu1|445031|estExtDG_fgenesh_newKGs_kg.C_3860014
jgi|Emihu1|369768|fgenesh_newKGs_kg.390__13__CX779085
33
jgi|Emihu1|445711|estExtDG_fgenesh_newKGs_kg.C_5000002
jgi|Emihu1|122419|fgeneshEH_pg.1261__3
jgi|Emihu1|254182|gm1.215400002
jgi|Emihu1|460878|estExtDG_Genemark1.C_88480001
34
8
References
1. Amino Acid Index: Download by anonymous FTP. (2015). aaindex1 [Data file].
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http://ieeexplore.ieee.org/xpl/login.jsp?tp=&arnumber=6845503&url=http%3A%2F%2Fi
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3D6845503
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