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Oligonucleotides for a Lentiviral Saturating Mutagenesis Screen

This page allows you to download all files for the ordering and the analysis of your oligonucleotide pool.

1 - Subpool barcodes: The minimum order is usually several thousand guides and it is cheaper to order more oligos. To reduce the cost per oligo, subsets can be tagged with a "barcode" (unrelated to Illumina sequencing index barcodes) so they can be selectively amplified from the pool.

Subpool barcode:

2 - PCR primers: Output file C includes two primers per target, for cleavage analysis with high-throughput sequencing. Primers are prefixed with Illumina Adapters. The forward primer prefix is TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG and the reverse prefix is GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG. These prefixes are already added to the primers in the Excel/Tab-sep tables.
Maximum amplicon length:     Primer Tm:

3 - Filters
Minimum specificity score:
A minimal value of 30 will remove only the guides in repeated regions. For screens, many researchers do not care a lot about off-targets. Increase this threshold if you want to more aggressively remove guides with many predicted off-targets.
Minimum Doench2016 efficiency score:
A minimal value of 10 will only remove the least efficient guides. Increase this if want to enrich more for predicted high efficiency guides.

4 - File format: Excel tables include a header with information how the oligonucleotides were constructed.
Text files have no header and are easier to process with other software.
Cleavage analysis files for Crispresso need to be in text format and are therefore not available as Excel files.
File format:

Output files:

  • A - Saturating Mutagenesis Oligonucleotides: the oligonucleotides to order from your Custom Oligonucleotide Array Supplier
  • B - Selection Analysis: for every oligo from A, its sequence and genome location. For CrisprSurf quantification
  • C - On-target sequencing primers: one forward and one reverse primer for every target from B in the input sequence
  • D - Cleavage Analysis: for each pair of primers from C, a table with the PCR amplicon and the guide. For CrispressoPooled, to analyze DNA cleavage induced by this guide
You can click the four buttons below and save all four files.

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