Technical data

5 Indexing and Sample Processing for Multiplexed Sequencing

Step 1B. Amplify the captured libraries with indexing primers containing 6-bp indexes 1–16

74 SureSelect

Target Enrichment System for Illumina Multiplexed Sequencing

Step 1B. Amplify the captured libraries with indexing primers

containing 6-bp indexes 1–16

In this step, the SureSelect-enriched DNA libraries are PCR amplified in

PCR reactions that include the appropriate indexing primer for each

sample.

This step uses the components listed in Table 41. Thaw then vortex to mix

the reagents listed below and keep on ice.

Prepare one indexing amplification reaction for each DNA library.

1 Determine the appropriate index assignments for each sample. See

Table 57 in the “Reference” chapter for sequences of the index portion

of the SureSelect PCR Primers Index 1 through Index 16 used to

amplify the DNA libraries in this step.

Use a different indexing primer for each sample to be sequenced in the

same lane.

Table 41 Reagents for post-capture indexing by PCR amplification

Kit Component Storage Location

5× Herculase II Reaction Buffer Herculase II Fusion DNA Polymerase kit*, –20°C

100 mM dNTP Mix (25 mM each dNTP) Herculase II Fusion DNA Polymerase kit

, –20°C

* Do not use the PCR Reaction Buffer or dNTP mix from any other kit.

Herculase II Fusion DNA Polymerase Herculase II Fusion DNA Polymerase kit, –20°C

SureSelect ILM Indexing Post-Capture Forward

PCR Primer

SureSelect Target Enrichment Kit ILM Indexing

Hyb Module Box 2, –20°C

SureSelect

Index reverse primers SureSelect XT Library Prep Kit ILM, –20°C

CAUTION

To avoid cross-contaminating libraries, set up PCR mixes in a dedicated clean area or

PCR hood with UV sterilization and positive air flow.