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

PROCEDURE - Solexa –Cluster Amplification

1) Load Samples to Flow Cell

8 Lanes are loaded onto the flow cell for simultaneous analysis

2. Attach DNA to Surface

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

3. Bridge Amplification

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

4. Fragments Become Double Stranded

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.

PROCEDURE - Solexa –Cluster Amplification

5. Double Stranded Molecules are Denatured

6. Amplification is Completed

PROCEDURE- Solexa Sequencing & Genome Analyzer

1. Determine 1st 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.

2. Image 1st Base 3. Determine 2nd Base

PROCEDURE- Solexa Sequencing & Genome Analyzer4. Image 2nd Base

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.

5. Sequence Read Continues Over Multiple Chemistry Cycles

6. Align and Map Data

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?

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