Gs titanium sequencing kit

Pyrosequencing of the amplicons was performed according to the manufacturer's protocol using the GS Junior System (Roche Diagnostics). Emulsion PCR, breaking, and bead enrichment were conducted using the GS Junior Titanium emPCR Kit, Lib-L emPCR Reagents, Lib-L Kit, Oil and Breaking Kit, and the Bead Recovery Reagents Kit according to the supplier's instructions (Roche Diagnostics). For emulsion PCR, we used a copy-per-bead ratio of 0.5. Enrichment of the DNA-carrying magnetic beads was accomplished using a magnetic particle collector (Invitrogen, Life Technologies). The quantity of the enriched beads was determined with the GS Junior Bead Counter (Roche Diagnostics). Finally, we loaded 100,000 to 500,000 beads onto the PicoTiterPlate (Roche Diagnostics). Sequencing was carried out according to standard Roche/454 protocols using the GS Titanium Sequencing Kit (Roche Diagnostics) and the GS Junior device.

The amplicon library was prepared according to the “Rapid Library Preparation Manual” (available on: http://454.com/downloads/my454/documentation/gs-junior/method-manuals/GSJuniorRapidLibraryPrepMethodManual_March2012.pdf) using multiplex identifiers (MIDs). The library was added to the emulsion PCR at a ratio of 0.2 molecules per bead, and the emulsion PCR amplification was performed as described in the “emPCR Amplification Method Manual–Lib-L” (available on http://454.com/downloads/my454/documentation/gs-junior/method-manuals/GSJunioremPCRAmplificationMethodManualLib-L_March2012.pdf). After a bead recovery and enrichment procedure, a picotiter plate was prepared. Approximately 500,000 enriched beads were loaded and sequenced according to the “Sequencing Method Manual” (http://454.com/downloads/my454/documentation/gs-junior/method-manuals/GSJuniorSequencingManual_Jan2013.pdf). Sequencing reactions were performed using a Roche 454 Junior Sequencer with GS Titanium Sequencing Kit according to the manufacturer's instructions (Roche Life Science).
Imaging and signal processing was competed using the GS Junior gsRunProcessor v3.0 full Processing, shotgun library pipeline.