title: Custom pangenomic 4 X 72 K Nimblegen array (eight strains of  Propionibacterium freudenreichii)
status: Public on Jun 13 2017
contact.city: Jouy-en-Josas
contact.first: Pierre
contact.last: Nicolas
contact.email: pierre.nicolas@inrae.fr
contact.address: INRAE - Domaine de Vilvert
contact.country: France
contact.institute: INRAE - Université Paris-Saclay
contact.phone: +33-1-3465-2894
organism: Propionibacterium freudenreichii
description: Propionibacterium freudenreichii strains CIRM121, CIRM122, CIRM129, CIRM134, CIRM139, CIRM456, CIRM514, and CIRM516.
  The design allows to monitor the expression of 2,740 genes (clusters) based on probing 27,546 distinct genomic regions (only 594 genes found in at least 1 of the 8 strains were not covered). On average, the expression of a gene was monitored via 10 distinct probe sequences (corresponding to 20 measures of intensity since each probe was duplicated).
  120412_Pfreundenreichii_VL_exp.ndf
  120412_Pfreundenreichii_VL_exp_X4.ncd
manufacture_protocol: 4 X 72K probes Nimblegen microarray. For the probe design we used the Oligowiz software (Wernersson and Nielsen, 2005) and a reference pangenome composed of a representative sequence for each gene cluster to build a repertoire of possible isothermal probes of length between 45 nt and 65 nt. A relatively high target melting temperature 90°C (+/- 5°C) was selected to account for the high G+C-content of Pf (67%). Then, we proceeded to the probe selection according to two criteria. First, we required at least 45 nt between the 5'-ends of two adjacent probes on the reference sequence (implying the overlap to be less than 20 nt). Second, we preferred probes corresponding to genomic regions without SNPs and discarded all probes whose sequences contained more than two mismatches between any of the 8 genomes and the reference pangenome. When SNPs existed, we included in the design a perfect match variant for each probe allele in the 8 genomes, and only perfect match variant was used to measure gene expression. For technical reasons, probes requiring more than 148 synthesis cycles were also filtered out. Finally, all probes were duplicated.