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RNA-Seq. An alternative to microarray. Grow cells or isolate tissue (brain, liver, muscle) Isolate total RNA Isolate mRNA from total RNA (poly A+ select) Fragment RNA Make and amplify cDNA Sequence the ends of the cDNA Map the sequences to the human genome

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rna seq

RNA-Seq

An alternative to microarray

steps
Grow cells or isolate tissue (brain, liver, muscle)

Isolate total RNA

Isolate mRNA from total RNA (poly A+ select)

Fragment RNA

Make and amplify cDNA

Sequence the ends of the cDNA

Map the sequences to the human genome

Count the number of sequence tags at each known gene (and at locations for which no gene is known)

Correct for background

Analyze the data

Steps
slide5

PROCEDURE - Solexa –Cluster Amplification

1) Load Samples to Flow Cell

2. Attach DNA to Surface

3. Bridge Amplification

8 Lanes are loaded onto the flow cell for simultaneous analysis

Single stranded DNA fragments bind randomly to the inside surface of the flow cell.

Unlabeled nucleotides and enzyme are added to initiate solid-phase bridge amplification.

slide6

PROCEDURE - Solexa –Cluster Amplification

4. Fragments Become

Double Stranded

5. Double Stranded Molecules

are Denatured

6. Amplification is Completed

The enzyme incorporates nucleotides to build double stranded bridges on the solid-phase substrate.

Denaturation leaves single-stranded template anchored to the substrate

Several million dense clusters of double stranded DNA are generated in each channel of the flow cell.

slide7

PROCEDURE- Solexa Sequencing & Genome Analyzer

1. Determine 1st Base

2. Image 1st Base

3. Determine 2nd Base

The first sequencing cycle is initiated by adding all 4 labeled reversible terminators, primers, and DNA polymerase to the flow cell

After laser excitation, an image of the emitted fluorescence from each cluster on the flow cell is captured.

The 2nd sequencing cycle is initiated by adding all 4 labeled reversible terminators and enzymes.

slide8

PROCEDURE- Solexa Sequencing & Genome Analyzer

4. Image 2nd Base

5. Sequence Read Continues Over Multiple Chemistry Cycles

6. Align and Map Data

After laser excitation, image data is collected like before. The identity of the 2nd base for each cluster is recorded.

35 cycles of sequencing are repeated to determine the sequence of bases in a given fragment a single base at a time.

Align data and map the sequences to the reference genome.

what are the similarities and differences
What can you learn with each one?

What can you learn from one but not from the other?

How is the primary data acquired?

How are systematic biases eliminated?

How do you normalize

How would you look for differential expression?

How would you cluster?

How can you combine data from multiple experiments?

Which is more sensitive?

What kinds of additional software do you need?

What are the similarities and differences?
references
Mortazavi A, Williams BA, McCue K, Schaeffer L, Wold B. Mapping and quantifying mammalian transcriptomes by RNA-Seq. Nature Methods (2008) 5: 621.

http://www.illumina.com/pages.ilmn?ID=203

References