Technical data

SureSelect

Target Enrichment System for Illumina Paired-End

Sequencing Library Protocol

Agilent Technologies

Indexing and Sample Processing for

Multiplexed Sequencing

Step 1A. Amplify the captured libraries with indexing primers containing

8-bp indexes A01–H12 70

Step 1B. Amplify the captured libraries with indexing primers containing

6-bp indexes 1–16 74

Step 2. Purify the amplified captured libraries using AMPure XP beads 77

Step 3. Assess indexed library DNA quantity and quality 79

Step 4. Quantify each index-tagged library by QPCR (optional) 83

Step 5. Pool samples for multiplexed sequencing 84

Guidelines for sequencing sample preparation and run setup 86

This chapter describes the steps to add index tags by amplification, and to

purify and assess quality and quantity of the captured libraries. Sample

pooling instructions are provided to prepare the indexed samples for

multiplexed sequencing.

Note that Step 1A and Step 1B are alternative protocols. Follow the

protocol appropriate for the set of indexing primers provided with your

kit. Use Step 1A if your kit includes the reconfigured 8-bp indexing

primers A01 to H12, provided in white-capped tubes (16 reaction kits) or

a blue plate (96 reaction kits). Use Step 1B if your kit includes the

original 6-bp indexing primers 1 to 16, provided in clear-capped tubes.