title: HMS/Somero-Mytilus-105K Agilent-v1.0 salinity stress
status: Public on Jan 10 2011
contact.city: Pacific Grove
contact.first: Brent
contact.middle: L
contact.last: Lockwood
contact.email: blockwoo@stanford.edu
contact.state: CA
contact.address: 120 Oceanview Blvd.
contact.department: Biology
contact.country: USA
contact.institute: Hopkins Marine Station of Stanford University
contact.phone: 831-655-6238
organism: Mytilus californianus
  Mytilus galloprovincialis
description: Agilent-019153
  
  To accurately compare the transcriptomes of Mytilus galloprovincialis and M. trossulus, we chose to develop a common microarray format that could be used for both species. This microarray design consisted of probe sequences generated from the out-group species, M. californianus. M. trossulus and M. galloprovincialis are approximately 7.5 million years divergent from M. californianus, yet only 3.5 million years divergent from each other (Seed, 1992). Therefore, heterologous hybridization to the microarray allowed us to compare transcriptional responses of M. galloprovincialis and M. trossulus without the inherent sequence biases that would result from a microarray that was designed from sequences of either M. galloprovincialis or M. trossulus. A limited number of sequences (556) from ESTs from M. galloprovincialis that matched M. californianus ESTs were included on the microarray to test for the effects of sequence mismatches. Only probes that performed well for both M. galloprovincialis and M. trossulus were used in our analyses.
  
  This is the same microarray platform as GPL9676 HMS/SomeroLab-Mytilus-105K array-v1.0, but the probe data are uploaded here separately due to the increased number of probes that had good hybridization in this study. This discrepancy was due to our stringent quality control that removed bad probes from the dataset and the fewer number of samples in this study (36 arrays in this study vs. 84 arrays in the previous study (Lockwood et al. 2010, Journal of Experimental Biology, 213, 3548-3558)).
  
  The Platform data table reflects a condensed representation of the array's replicate features. The full array layout representing all of the individual features is linked as a supplementary file at the foot of this record.
manufacture_protocol: Approximately 26,000 publicly available ESTs from M. californianus (ES7325872 - ES738966; ES408175 - ES387463;(Gracey et al., 2008)) and M. galloprovincialis (AJ623313  - AJ626468; AJ516092 - AJ516921; (Venier et al., 2003)). SSH to enrich for transcripts expressed in response to various stressors.
  
  Sequences for each species were separately screened for vector contamination, clustered, assembled, and loaded into a PostgresQL database using the PartiGene bioinformatics pipeline (Parkinson et al., 2004), yielding a total of 12,961 and 1,688 putative gene transcripts (clusters) for M. californianus and M. galloprovincialis sequences, respectively. Clusters were annotated for identity against the UniRef 90 protein database using NCBI Blast, with an e-value cutoff of 10-8. Clusters were also annotated for gene ontology (GO), enzyme classification (ECC), and gene pathway (KEGG) using the program annot8r (Schmid and Blaxter, 2008), again with an e-value cutoff of 10-8.  Long oligo (60-mer) probes were designed against the M. californianus clusters using the probe design program YODA (Nordberg, 2005) under default parameters, with a maximum of 5 probes per cluster. This yielded 43,969 total unique probes to 11,506 clusters, with a mean of 2.6 probes per cluster. To help control for the effects of interspecific sequence variation on probe performance, the above probes were compared to M. galloprovincialis clusters using BLAST. Probes designed to the M. californianus library that successfully aligned with the smaller M. galloprovincialis library had counterparts designed using the M. galloprovincialis sequence. This resulted in 556 matched pairs of probes, with a mean number of 4.6 mismatches per probe. Altogether, there were 44,524 unique probes that were duplicated or triplicated randomly to fill a 105k-feature microarray and in-situ synthesized by Agilent Technologies, Inc. (Santa Clara, CA, USA).