6.4. Sequencing sgRNA Inserts or Barcodes

The Second Round nested PCR primers contain sequences (P5 and P7) complementary to the sequences of the immobilized primers necessary for generating amplification clusters on any of Illumina’s NGS Instruments. In addition, if you are using a Cellecta NGS Prep Kit, the NRev Index Primers add sample-specific index sequences which allow you to combine and multiplex up to 24 samples on a single flow cell or lane. The primers were designed with the NextSeq instrument in mind, but they are compatible with other Illumina instruments as well.

Note: Please see the NGS Cassette Diagram for the specific Cellecta library or the Product Certificate for the NGS Prep Kit you purchased for information on compatibility with HiSeq Single-Read (SR) and Paired-End (PE) Reagent Kits.

*IMPORTANT: For certain libraries, such as sgRNA and dual-sgRNA, the Cellecta 6-nt Index should be read using Read 2, not in the Illumina Index 1. Please check the Product Certificate for the appropriate LNGS Kit for more information.

The number of sequencing cycles (read length) required depends on the sgRNA or barcode length. Please refer to your library information (NGS Cassette Diagram for the specific Cellecta library or the Product Certificate for the NGS Prep Kit).

Guidelines for preparing samples for sequencing:

• Combine together equal amounts (each adjusted to 10 nM) of experimental and positive control samples to be sequenced together. Importantly, each sample in the combined pool needs to have a unique index sequence.

!We strongly recommend that you DO NOT combine Cellecta samples with samples of different origin on a single flow cell or lane. It may cause sequencing errors.

Note: The number of samples which can be sequenced in one lane or flow cell is based on the complexity of the pooled library and expected total number of reads generated with the Illumina instrument. You will need to determine how many target reads you want per sample based on your experiment.

• Prepare the samples for NGS by following the Illumina “Denature and Dilute Libraries Guide” specific for your system.
  • NextSeq 500: https://support.illumina.com/downloads/nextseq-500-denaturing-diluting-libraries-15048776.html
  • HiSeq: https://support.illumina.com/downloads/hiseq-denature-dilute-libraries-guide-15050107.html

• Spike in 5-15% of the PhiX Control to the pooled indexed sample to increase the nucleotide diversity.

• Mix Cellecta’s Seq NGS Primer with the Illumina primer mix (for the NextSeq, spike in the primer at the appropriate cartridge position—usually at Read 1). For indexing multiple samples in a flow cell or lane using a Cellecta NGS Prep Kit, also add the Index NGS Primer directly to the appropriate well (i.e., spike in either with the Illumina index primer or the reverse sequencing primer; see Note below). Each primer should have a final concentration of 300 nM for the NextSeq, or 500 nM for the HiSeq. We recommend measuring the final volume of the primer mix in each well to ensure accurate spiked-in primer concentrations.

Note: Depending on the way the amplification and sequencing primers were designed, the position of the Seq NGS and Index NGS primers may vary. Refer to the supplementary information (Product Certificate or NGS Cassette Diagram file) provided for the specifics on how to set up the NGS Primers for your specific library.

• Specify the appropriate number of cycles to sequence the complete sgRNA or barcode region (please refer to Product Certificate or NGS Cassette Diagram file), and specify the indicated number of cycles to read the 6-base Index sequence if using a Cellecta NGS Prep Kit.