Sequencing Transcriptomes Do Me a SOLiD

Overview – Library Construction RNA ◦Isolate & Bioanalyze ◦rRNA Depletion ◦Fragment ◦Bioanalyze Amplified Library Construction ◦Hybridize & Ligate RNA ◦Reverse Transcription ◦cDNA Size Selection/Amplification RNA ◦Isolate & Bioanalyze ◦rRNA Depletion ◦Fragment ◦Bioanalyze Amplified Library Construction ◦Hybridize & Ligate RNA ◦Reverse Transcription ◦cDNA Size Selection/Amplification

RNA ◦Isolate & Bioanalyze ◦rRNA Depletion ◦Fragment ◦Bioanalyze Amplified Library Construction ◦Hybridize & Ligate RNA ◦Reverse Transcription ◦cDNA Size Selection/Amplification RNA ◦Isolate & Bioanalyze ◦rRNA Depletion ◦Fragment ◦Bioanalyze Amplified Library Construction ◦Hybridize & Ligate RNA ◦Reverse Transcription ◦cDNA Size Selection/Amplification Overview – Library Construction

Emulsion PCR (ePCR) Emulsion break/bead wash Enrich templated beads Modify 3’ ends Emulsion PCR (ePCR) Emulsion break/bead wash Enrich templated beads Modify 3’ ends Overview – Templated Bead Prep

Library Construction RNA – Isolate -> Bioanalyzer

Library Construction RNA – Bioanalyzer

Library Construction RNA – Total, mRNA, Fragmented

Library Construction RNA – Total, mRNA, Fragmented

Library Construction Fragmented RNA – Ligate, Reverse Transcribe Adaptor ligated to fragmented RNA Primer binding site during cDNA amplification Adaptor ligated to fragmented RNA Primer binding site during cDNA amplification

Library Construction cDNA – Size Selection 6% TBE-Urea gel 180V for ~17 mins 6% TBE-Urea gel 180V for ~17 mins bp range excised Cut vertically 4 equal pieces (~1x6mm) In-gel PCR Only cycles Primers 1 & 2 bind to adaptors Barcode? bp range excised Cut vertically 4 equal pieces (~1x6mm) In-gel PCR Only cycles Primers 1 & 2 bind to adaptors Barcode?

Library Construction Amplified cDNA – Yield, Size Distribution

Library Construction Amplified cDNA – Yield, Size Distribution

Templated Bead Prep Amplified cDNA – ePCR pM Template (~60-180pg/uL) Empirically determined  pM Template (~60-180pg/uL) Empirically determined 

Templated Bead Prep Amplified cDNA – ePCR Before PCR

Templated Bead Prep Amplified cDNA – ePCR

Templated Bead Prep Amplified cDNA – ePCR

Templated Bead Prep Amplified cDNA – ePCR After PCR

Templated Bead Prep Amplified cDNA – ePCR Centrifuge

Templated Bead Prep ePCR – Emulsion Break

Templated Bead Prep ePCR – Emulsion Break/Bead Counts

Templated Bead Prep Bead Enrichment Templated Beads + P2 Enrichment Beads on Glycerol Cushion

Templated Bead Prep Bead Enrichment Low templated bead recovery at interphase.

Templated Bead Prep Bead Enrichment Ideal templated bead recovery at interphase.

Templated Bead Prep Bead Enrichment 3’ Modification Bead Counts # Templated Beads ( million = Good) % Templated Beads (<20% = Good) 3’ Modification Bead Counts # Templated Beads ( million = Good) % Templated Beads (<20% = Good)

SOLiD Run Sequencing

SOLiD Run Sequencing Bead Depostion Chambers

SOLiD Run Sequencing

SOLiD Run Sequencing - Reagents

SOLiD Run Sequencing – Focus Beads WFA Run (~5500 beads/panel) WFA Run (~5500 beads/panel) Sequencing Run (~220,000 beads/panel) Sequencing Run (~220,000 beads/panel)

SOLiD Run Sequencing - Cycling

SOLiD Run Sequencing - Interrogation

SOLiD Run Sequencing – Satay Plots Quality Check Spectral Purity Signal Intensity Quality Check Spectral Purity Signal Intensity

SOLiD Run Sequencing – Satay Plots Cycle #1 Cycle #10

SOLiD Run Sequencing – Cycle Scans

SOLiD Run Sequencing – Exposure Time Quality Check Focus Reagents Quality Check Focus Reagents

SOLiD Run Sequencing – Color Balance Quality Check Library Prep (Bias?) Reagents Quality Check Library Prep (Bias?) Reagents

SOLiD Run Sequencing – Heat Maps Quality Check Bead Count Deposition Bead Signal Image Signal Background Quality Check Bead Count Deposition Bead Signal Image Signal Background

SOLiD Time cDNA Library (RNA to cDNA) = 3 days ePCR (ePCR to WFA) = 2 days WFA = 1 day SOLiD Run = 7 days Total Time = 13 days Nucleotides/Day* = 1.54x10^8 bp/day * - 40M reads of 50bp each

SOLiD Money mRNA Library Construction = $225 = $20 Bead Prep = $350 SOLiD Reagents = $500 (?) Total Cost = ~$1200** Cost/Nucleotide* = 6x10^-7 dollars/bp * - 40M reads of 50bp each ** - Includes miscellaneous/incidentals

That’s SOLiD!