Your screening samples are unique, so are the Pooled Screening Indexing PCR Primers.
Thanks to next-generation sequencing (NGS), pooled screening is made possible to provide an efficient and economical method to screen for large numbers of genes that are critical for the biological system in your research. However, for a scientist moving into the pooled screening technology, sometimes the terminology can be a bit confusing. Here we have gathered the most common questions about our Indexing PCR and Sequencing Primer kit, so you will be sounding like an expert in no time. While we are focusing here on our sgRNA indexing primers, this information also applies to SMARTvector Indexing Primers.
What is the role of the Edit-R Pooled sgRNA Indexing PCR Primers?
The Edit-R Pooled sgRNA Indexing PCR Primers are Illumina™-adapted for identification of single guide RNA (sgRNA) targeting sequences from genomic DNA (gDNA) during the high-throughput sequencing step. We offer one universal forward PCR primer, 12 reverse index PCR primers in kit A, and 12 reverse index primers in kit B, each with unique index sequences. The forward PCR primer aligns with the pro-viral sequence upstream of the gene-specific Edit-R sgRNA sequence. The reverse Index PCR primer aligns with the pro-viral sequence downstream of the gene-specific Edit-R sgRNA sequence and has a built-in index sequence that is incorporated into the amplicon sequence during the amplification step. Each reverse index primer has its own unique index sequence which serves as an identifier to allow mixing, or multiplexing, in the same sequencing lane, of amplicons that are amplified with different indices. Most typically these are used to identify separate cell populations or conditions within the screen. Being able to analyze more than one sample/condition/replicate per lane can help minimize the cost and run time of the sequencing procedure.
What is the function of the two sequencing primers?
At the sequencing step, the first round of sequencing is performed by the Edit-R pooled sgRNA Read 1 sequencing primer (purple color in Figure 1), which recognizes the 5' fragment of the amplicon (amplified from the pro-viral sequence upstream of the sgRNA). Specific sgRNA sequence will be read out at this step.The second round of sequencing is performed by the Edit-R Pooled sgRNA Index Read sequencing primer (yellow color in Figure 1) representing two different index primers (primers in partial red and green in Figure 1) that recognizes the amplified pro-viral sequence downstream of the sgRNA. This primer reads out the index sequences that would have been incorporated into the amplicon at the PCR step. These steps combined allow researchers to distinguish outcome from one condition/treatment from another to ensure accurate target gene identification.
Index primers diagram
Where can I find the Edit-R pooled sgRNA index sequences?
Index sequences can be found in the Edit-R pooled screening Technical Manual.
Can I use sequencing primers from a different vendor?
The Edit-R Pooled sgRNA Indexing PCR Primers were specifically designed and are validated to work with the vector used in the library. While other reagents might be successful after considerable careful optimization, we would not generally recommend using the universal sequencing primers provided by the sequencing facility.
Can I multiplex, or share lanes, with samples from another researcher?
We do not recommend mixing your sample with others that were amplified by primers from a different vendor, as that may cause interference and result in low read quality.
Does the SMARTvector Indexing PCR and Sequencing Primer Kit work in the same way? Can I use these for my CRISPR pooled screen?
The SMARTvector Indexing PCR and Sequencing Primer Kit works similarly for the SMARTvector lentiviral shRNA/shMIMIC Lentiviral microRNA Pooled Libraries. However, the SMARTvector Indexing PCR and Sequencing Primer Kit cannot be used for CRISPR pooled screen as the pro-viral sequences from SMARTvector is different from the Edit-R sgRNA.
Indexing PCR and Sequencing Primer kit – the effective facilitator for your successful pooled screening
The convenience and cost-effectiveness of pooled screening libraries is further enhanced by the use of the Indexing PCR and Sequencing Primer kit. The primer kit allows the researcher to effortlessly transition from PCR amplification to NGS and to multiplex different samples/conditions/replicates during sequencing. With the high-quality sgRNA/shRNA pools and optimized primer kits from Horizon, you are equipped with the best tools to achieve a successful pooled screening.
Author: Annie Zhang Bargsten, M.D., Ph.D. | Scientific Support Specialist 2
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