Statistics for K-mer Based Splicing Event Analysis

Data Learner Miner Practitioner

Ruofei Du, Hao Li, Hui Miao, Shangfu Peng

Alternative Splicing Events

Image from: "Alternative Splicing Event" Wikipedia: The Free Encyclopedia. Wikimedia Foundation, Inc. 2 Apr. 2014. <http://en.wikipedia.org/wiki/Alternative_splicing>

● Alternative splicing is used to describe

any case in which a primary transcript

can be spliced in more than one pattern

to generate multiple and distinct

mRNAs.

● 5 traditional basic modes; most

common: exon skipping.

● It is a widespread mechanism for

generating protein diversity and

regulating protein expression.

● Improve

understanding of

cell

differentiation

and classify

disease types

Image from: Sammeth, Michael, Sylvain Foissac, and Roderic Guigó. "A General Definition and Nomenclature for Alternative Splicing Events." PLoS Computational Biology 4.8 (2008): e1000147.

Alternative Splicing Events

● Different species tend to have different splicing event patterns.

● Different splicing events also indicates the abnormal cells activities, such as cancer

Image from: Sammeth, Michael, Sylvain Foissac, and Roderic Guigó. "A General Definition and Nomenclature for Alternative Splicing Events." PLoS Computational Biology 4.8 (2008): e1000147.

Abundance Estimation for Alternative Splicing Events

● Given RNA-Seq samples, estimate the abundance and the relative proportion of every alternative transcription path

Image from: Hu, Yin, et al. "DiffSplice: the genome-wide detection of differential splicing events with RNA-seq." Nucleic acids research 41.2 (2013): e39-e39.

Abundance Estimation for Isoforms

● The Standard Paradigmo Read alignment step can be very computationally

intensive.

● Sailfisho Far faster than the standard paradigm

o Replace the step of read mapping with the much

faster and simpler process of k-mer counting

Sailfish: Alignment-free Isoform Quantification from RNA-seq Reads using Lightweight Algorithms Rob Patro, Stephen M. Mount, and Carl Kingsford. Manuscript Submitted (2013) http://www.cs.cmu.edu/~ckingsf/class/02714-f13/Lec05-sailfish.pdf

K-mer

● A fixed sized (K) sequence

Sailfish: Alignment-free Isoform Quantification from RNA-seq Reads using Lightweight Algorithms Rob Patro, Stephen M. Mount, and Carl Kingsford. Manuscript Submitted (2013) http://www.cs.cmu.edu/~ckingsf/class/02714-f13/Lec05-sailfish.pdf

A

C

G

T

AA AC AG AT

CA CC CG CT

GA GC GG GT

TA TC TG TT

● A string of length N contains N-K+1 k-mers

● One can build K-mer index to represent a string

7-mer iD N

ATTCGAC 1 1

TTCGACA 2 1

TCGACAG 3 1

...

1-mer 2-mer

Sailfish Workflow

● Indexingo Build K-mer index for known

isoform transcripts

Sailfish: Alignment-free Isoform Quantification from RNA-seq Reads using Lightweight Algorithms Rob Patro, Stephen M. Mount, and Carl Kingsford. Manuscript Submitted (2013)

● Quantificationo Counts the number of times

each K-mer occurs in the reads.

o Estimating abundances via an EM algorithm

Sailfish Workflow: Indexing

● Perfect Hashing

http://www.cs.cmu.edu/~ckingsf/class/02714-f13/Lec05-sailfish.pdf

Domain(K-mer) Range([0,|D|-1])

Sailfish Workflow: Quantification

2.K-mer Allocation to Transcripts

http://www.cs.cmu.edu/~ckingsf/class/02714-f13/Lec05-sailfish.pdf

1. Read Data K-mer Counting

Our Proposal

● We propose to investigate the scalable statistic method using k-mer and k-mer index to estimate abundance of alternative splicing events.

● We will focus on the most frequent event type:Exon Skipping Evento other event types can

be extended naturally

Shen, Shihao, et al. "MATS: a Bayesian framework for Flexible Detection of Differential Alternative Splicing from RNA-Seq Data." Nucleic Acids Research 40.8 (2012): e6

(1) (2) (3)

● Variables for abundance:

● Build k-mer index for a specific gene: e.g. A B C D E

● On reads part, aggregated k-mer counts like Sailfish

● Use EM to do maximum likelihood estimation

Class I: Each exon i

Class II: Each exon-exon junction (non-spliced)

Class III: Each spliced junction

Initial Idea

Exon A, B, C, D, E

Non-spliced junction AB, BC, CD, DE

Spliced junction AC, BD, CE

Advantage

● Do not require to know the Isoform space.

● Replace the step of read mapping, and provide a faster

approach for splicing event analysis.

Thank you

Questions

1. The drawback of the straightforward method: get the Pi of each Isoform using

EM first, and then calculate the frequency of events.

2. Why we have to use EM, why not solve equations?

3. Require to know the frequency of the five events?