An Introduction to BioinformaticsToolsPart 1: Golden Rules of Bioinformatics

Leighton Pritchard and Peter Cock

On Confidence

“Ignorance more frequently begets confidence than doesknowledge: it is those who know little, not those who know much,who so positively assert. . .”- Charles Darwin

Table of Contents

Rule 0

Rule 1

Rule 2

Rule 3

Conclusions

Zeroeth Golden Rule of Bioinformatics

• No-one knows everything about everything - talk to people!• local bioinformaticians, mailing lists, forums, Twitter, etc.

• Keep learning - there are lots of resources

• There is no free lunch - no method works best on all data

• The worst errors are silent - share worries, problems, etc.

• Share expertise (see first item)

Table of Contents

Rule 0

Rule 1

Rule 2

Rule 3

Conclusions

Subgroups

• You are in group A, B, C or D - this decides your dataset:expnA.tab, expnB.tab, expnC.tab, expnD.tab

• You will use R at the command-line to analyse your data

The biological question

• Your dataset expn?.tab describes (log) expression data fortwo genes: gene1 and gene2

• Expression measured at eleven time points (including control)

• Q: Are gene1 and gene2 genes coregulated?

• How do we answer this question?

Reformulating the biological question

• Q: Are gene1 and gene2 genes coregulated?

• A: We cannot determine this from expression data alone

• Reformulate the question:

• NewQ: Is there evidence that gene1 and gene2 expressionprofiles are correlated?(is expression gene1 ∝ gene2)

• How do we answer this new question?

Reformulating the biological question

• Q: Are gene1 and gene2 genes coregulated?

• A: We cannot determine this from expression data alone

• Reformulate the question:

• NewQ: Is there evidence that gene1 and gene2 expressionprofiles are correlated?(is expression gene1 ∝ gene2)

• How do we answer this new question?

Starting the analysis

• Change directory to where Exercise 1 data is located, andstart R.

1 $ cd ../../ data/ex1_expression/

2 $ R

Load and inspect data in R

1 > data = read.table("expnA.tab", sep="\t", header=TRUE)

2 > head(data)

3 gene1 gene2

4 1 10 8.04

5 2 8 6.95

6 3 13 7.58

7 4 9 8.81

8 5 11 8.33

9 6 14 9.96

Load and inspect data in R

1 > mean(data$gene1)

2 [1] 9

3 > mean(data$gene2)

4 [1] 7.500909

5 > sd(data$gene1)

6 [1] 3.316625

7 > sd(data$gene2)

8 [1] 2.031568

9 > cor(data)

10 gene1 gene2

11 gene1 1.0000000 0.8164205

12 gene2 0.8164205 1.0000000

Results

measure expnA expnB expnC expnD

mean(gene1) 9mean(gene2) 7.5

sd(gene1) 3.3sd(gene2) 2.0cor(data) 0.816

Results

measure expnA expnB expnC expnD

mean(gene1) 9 9 9 9mean(gene2) 7.5 7.5 7.5 7.5

sd(gene1) 3.3 3.3 3.3 3.3sd(gene2) 2.0 2.0 2.0 2.0cor(data) 0.816 0.816 0.816 0.816

• r = 0.816(P < 0.005) in every experiment

• Can we conclude that gene1 and gene2 are coexpressed ineach experiment?

Results

measure expnA expnB expnC expnD

mean(gene1) 9 9 9 9mean(gene2) 7.5 7.5 7.5 7.5

sd(gene1) 3.3 3.3 3.3 3.3sd(gene2) 2.0 2.0 2.0 2.0cor(data) 0.816 0.816 0.816 0.816

• r = 0.816(P < 0.005) in every experiment

• Can we conclude that gene1 and gene2 are coexpressed ineach experiment?

Plot the data in R

1 > plot(data)

Always plot the data

Which gene pairs are coexpressed?

Always plot the data

Is the matrix of (Pearson) correlation values potentially misleading?

1 > data = anscombe

2 > cor(data)[1:4 ,5:8]

3 y1 y2 y3 y4

4 x1 0.8164205 0.8162365 0.8162867 -0.3140467

5 x2 0.8164205 0.8162365 0.8162867 -0.3140467

6 x3 0.8164205 0.8162365 0.8162867 -0.3140467

7 x4 -0.5290927 -0.7184365 -0.3446610 0.8165214

Sometimes real correlation doesn’tmean anything

First Golden Rule of Bioinformatics

• Always inspect the raw data (trends, outliers, clustering)

• What is the question? Can the data answer it?

• Communicate with data collectors! (don’t be afraid ofpedantry)

• Who? When? How?• You need to understand the experiment to analyse it (easier if

you helped design it).• Be wary of block effects (experimenter, time, batch, etc.)

Table of Contents

Rule 0

Rule 1

Rule 2

Rule 3

Conclusions

Exercise 2

• You are in group A, B, C or D - this decides your databasedbA, dbB, dbC, dbD

• You will use BLAST at the command-line to analyse your data

• You will use script at the command-line to record your work

Exercise 2

• Start recording your actions by entering script at thecommand line

1 $ script

2 Script started , output file is typescript

Exercise 2

• Change directory to the ex2 blast directory

• Run BLAST with the appropriate database

• Exit script

1 $ cd ../ ex2_blast

2 $ blastp -num_alignments 1 -num_descriptions 1 -query query.fasta -db dbA

3 $ exit

4 exit

5 Script done , output file is typescript

Exercise 2

• You can view the typescript file with cat

1 $ cat typescript

2 Script started on Fri May 9 10:45:12 2014

3 lpritc@lpmacpro:$ cd ../ ex2_blast

4 [...]

Exercise 2

Query= query protein sequence

Length=400

Score

Sequences producing significant alignments: (Bits)

PITG_08491T0 Phytophthora infestans T30-4 choline transporter-l... 34.3

> PITG_08491T0 Phytophthora infestans T30-4 choline transporter-like

protein (441 aa)

Length=486

Score = 34.3 bits (77), Method: Compositional matrix adjust.

Identities = 22/69 (32%), Positives = 38/69 (55%), Gaps = 4/69 (6%)

Query 106 EVILPMMYQFALKPSFADVINDYKPYSKHTAGVSDQELKGEATTWMLADKNSRMKAFLSQ 165

E+++PM+Y L F ++ Y P HTA ++ EL+G T ++A+ S + F ++

Sbjct 40 ELMVPMLYSLYLVVLFHLPVSAYYP---HTASMTAHELQGAVITILVAETPSIIIQF-AK 95

Query 166 IKTKSNSSE 174

T SN S+

Sbjct 96 CHTSSNISQ 104

Exercise 2

• What is a reasonable E-value threshold to call a ’match’?• 1e-05, 0.001, 0.1, 10?

dbA dbB dbC dbD

E-value

Exercise 2

• What is a reasonable E-value threshold to call a ’match’?• 1e-05, 0.001, 0.1, 10?

dbA dbB dbC dbD

E-value 0.45 0.002 4e-06 0.019

• Five orders of magnitude difference in E-value, depending ondatabase choice - Why?

Exercise 2

• E-values depend on database size

• Bit score and alignment do not depend on database size

dbA dbB dbC dbD

E-value 0.45 0.002 4e-06 0.019Bit score 34.3 34.3 34.3 34.3

Sequences 100,001 501 1 5,001Letters 48,650,486 210,866 486 2,066,510

Exercise 2

• E-values differ, but the query matches a cholinetransporter-like protein quite well. . .

• Doesn’t it?

• After all, a biological match is a biological match. . .

• Isn’t it?

Exercise 2

• E-values differ, but the query matches a cholinetransporter-like protein quite well. . .

• Doesn’t it?

• After all, a biological match is a biological match. . .

• Isn’t it?

Exercise 2

Query= query protein sequence

Length=400

Score E

Sequences producing significant alignments: (Bits) Value

PITG_08491T0 Phytophthora infestans T30-4 choline transporter-l... 34.3 4e-06

> PITG_08491T0 Phytophthora infestans T30-4 choline transporter-like

protein (441 aa)

Length=486

Score = 34.3 bits (77), Expect = 4e-06, Method: Compositional matrix adjust.

Identities = 22/69 (32%), Positives = 38/69 (55%), Gaps = 4/69 (6%)

Query 106 EVILPMMYQFALKPSFADVINDYKPYSKHTAGVSDQELKGEATTWMLADKNSRMKAFLSQ 165

E+++PM+Y L F ++ Y P HTA ++ EL+G T ++A+ S + F ++

Sbjct 40 ELMVPMLYSLYLVVLFHLPVSAYYP---HTASMTAHELQGAVITILVAETPSIIIQF-AK 95

Query 166 IKTKSNSSE 174

T SN S+

Sbjct 96 CHTSSNISQ 104

Exercise 2

• Sequence accessions (PITG ?????T0) are correct in thedatabases

• Sequence functional descriptions are randomly shuffled:lengths do not match in BLAST output

• dbA contains only three different sequences: two are repeated50,000 times

• query.fasta is random sequence, not a real protein

• Shuffled from all P. infestans proteins• No nr or PFam matches

Exercise 2

• Sequence accessions (PITG ?????T0) are correct in thedatabases

• Sequence functional descriptions are randomly shuffled:lengths do not match in BLAST output

• dbA contains only three different sequences: two are repeated50,000 times

• query.fasta is random sequence, not a real protein

• Shuffled from all P. infestans proteins• No nr or PFam matches

Exercise 2

• Sequence accessions (PITG ?????T0) are correct in thedatabases

• Sequence functional descriptions are randomly shuffled:lengths do not match in BLAST output

• dbA contains only three different sequences: two are repeated50,000 times

• query.fasta is random sequence, not a real protein

• Shuffled from all P. infestans proteins• No nr or PFam matches

Exercise 2

• Sequence accessions (PITG ?????T0) are correct in thedatabases

• Sequence functional descriptions are randomly shuffled:lengths do not match in BLAST output

• dbA contains only three different sequences: two are repeated50,000 times

• query.fasta is random sequence, not a real protein• Shuffled from all P. infestans proteins• No nr or PFam matches

Second Golden Rule of Bioinformatics

• Do not trust the software: it is not an authority• Software does not distinguish meaningful from meaningless

data• Software has bugs• Algorithms have assumptions, conditions, and applicable

domains• Some problems are inherently hard, or even insoluble

• You must understand the analysis/algorithm

• Always sanity test

• Test output for robustness to parameter (including data)choice

Table of Contents

Rule 0

Rule 1

Rule 2

Rule 3

Conclusions

Exercise 3

• Rule: If there is a vowel on one side of the card, there mustbe an even number on the other side.

• Which cards must be turned over to determine if this rule (ifa card shows a vowel on one face, the opposite face is even)holds true?

Exercise 3

This is the Wason Selection Task

• If you chose E and 4

• You are in the typical majority group• You are not correct• You have been a victim of confirmation bias (System 1

thinking)

• If you chose E and 7

• Congratulations!• Your choice was capable of falsifying the rule.

Exercise 3

This is the Wason Selection Task

• If you chose E and 4• You are in the typical majority group• You are not correct• You have been a victim of confirmation bias (System 1

thinking)

• If you chose E and 7

• Congratulations!• Your choice was capable of falsifying the rule.

Exercise 3

This is the Wason Selection Task

• If you chose E and 4• You are in the typical majority group• You are not correct• You have been a victim of confirmation bias (System 1

thinking)

• If you chose E and 7

• Congratulations!• Your choice was capable of falsifying the rule.

Exercise 3

This is the Wason Selection Task

• If you chose E and 4• You are in the typical majority group• You are not correct• You have been a victim of confirmation bias (System 1

thinking)

• If you chose E and 7• Congratulations!• Your choice was capable of falsifying the rule.

Exercise 3

Rule: If there is a vowel on one side of the card, there must be aneven number on the other side.

Card Outcome Rule

EEven Can be true even if rule falseOdd violated

KEven naOdd na

4Vowel Can be true even if rule false

Consonant na

7Vowel violated

Consonant na

Exercise 3

• This is equivalent to functional classification, e.g:

• Rule: If there is a CRN/RxLR/T3SS domain, the protein mustbe an effector.

Exercise 3

• Confirmation Bias (Wason Selection Task)• An uninformative experiment is performed• http://en.wikipedia.org/wiki/Wason_selection_task

• Affirming the Consequent (a related formal fallacy)

1. If P, then Q2. Q3. Therefore, P

• Experimental results are misinterpreted• http:

//en.wikipedia.org/wiki/Affirming_the_consequent

Third Golden Rule of Bioinformatics

• Everyone has expectations of their data/experiment• Beware cognitive errors, such as confirmation bias!• System 1 vs. System 2 ≈ intuition vs. reason

• Think statistically!• Large datasets can be counterintuitive and appear to confirm a

large number of contradictory hypotheses• Always account for multiple tests.• Avoid “data dredging”: intensive computation is not an

adequate substitute for expertise

• Use test-driven development of analyses and code• Use examples that pass and fail

Table of Contents

Rule 0

Rule 1

Rule 2

Rule 3

Conclusions

In Conclusion

• Always communicate!• worst errors are silent

• Don’t trust the data• formatting/validation/category errors - check!• suitability for scientific question

• Don’t trust the software• software is not an authority• always benchmark, always validate

• Don’t trust yourself• beware cognitive errors• think statistically• biological “stories” can be constructed from nonsense