Sequence Data that never made it


Over the past several years our lab has experimented with a variety of high-throughput sequencing platforms with the ultimate goal of addressing important scientific questions. On a few occasions we have been successful. Some of our early work included Roche 454 GS-FLX pyrosequencing used to examine phenotypic plasticity in lake trout (FREDERICK 2010), identify single nucleotide polymorphisms in salmon (E. 2011) and develop genomic resources for Pacific herring (B. 2012).

  • Morera D, Roher N, Ribas L, Balasch JC, Doñate C, Callol A, Boltaña A, Roberts SB, Goetz G, Goetz FW, Mackenzie SA. (2011) RNA-Seq reveals an integrated immune response in nucleated erythrocytes. PLoS ONE 6(10): e26998. doi:10.1371/journal.pone.0026998

  • Gavery MR* and Roberts SB. (2012) Characterizing short read sequencing for gene discovery and RNA-Seq analysis in Crassostrea gigas. Comparative Biochemistry and Physiology Part D: Genomics and Proteomics, 7:2 94-99 June 2012. doi:10.1016/j.cbd.2011.12.003

  • Burge CA, Douglas N, Conti-Jerpe I, Weil E, Roberts SB, Friedman CS, CD Harvell. (2012) Friend or foe: the association of Labyrinthulomycetes with the Caribbean sea fan, Gorgonia ventalina. Diseases of Aquatic Organisms. 101:1-12 doi:10.3354/dao02487

  • Gavery MR, Roberts SB. (2013) Predominant intragenic methylation is associated with gene expression characteristics in a bivalve mollusc. PeerJ 1:e215 doi:10.7717/peerj.215

However there are a number of efforts that have failed early on (ie poor read quality) or just never gained enough momentum to see the light of the peer-review system. This paper is a final attempt to share these datasets in a manner where they have the potenial, however slim, to be useful to someone. We will not be providing fluffy background or in-depth over-analysis but strive to offer a sound data description. This current work will be organized by target species, which will often correspond to project.

Species include

  • Vibrio tubiashii
  • Olympia oyster (Ostrea lurida)
  • Yellow Perch (Perca flavescens)
  • Pacific herring (Clupea pallasii)
  • Abalone / Withering Syndrome (WS)
  • Hard clam (Mercenaria mercenaria)

Vibrio tubiashii

According to the our records the first library we ever sequenced was from Vibrio tubiashi. The experiment compared transcriptomic changes in V.tubiashii exposed to autoclaved C.gigas and those that were not. Samples were incubated in sterile seawater (with or without autoclaved C.gigas) for 24hrs. at room temperature, at a concentration of 9.975x10^11 CFU/mL. After 24hrs., V.tubiashii concentrations were calculated and two 50mL volumes were collected from each treatment. Cells were pelleted, supernatant removed and the pellets were stored @ -80C.

Total RNA was isolated from the control (5.63 x 10^11 CFU) and exposed (1.835 x 10^12 CFU) V.tubiashii samples using 10mL of TriReagent. The remainder of the manufacturer's protocol was scaled appropriately. Ribosomal RNA (rRNA) was removed from each sample using the MICROBExpress Kit (Ambion), accoriding to the manufacturer's protocol. Removal of rRNA from each sample was verified via formaldehyde-HEPES agarose gel and compared with untreated total RNA from each sample. Submitted just the C.gigas-exposed V.tubiashii mRNA for single-end Illumina sequencing at the High Throughput Genomics Unit (HTGU; University of Washington).

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As part of a Saltonstall-Kennedy Grant funded project entitled "Ocean acidification and emerging diseases in the Pacific Northwest" genomic libraries were generated and sequenced. Two different strains were partially sequenced in an effort to learn what genetic differences might be associated with different phenotypes (ie growth) under ocean acidification conditions.

The next occurance of this taxa was not until years later with two genomic DNA libraries: RE22 and ATCC 19106. These were SOLiD libraries sequenced in early 2011.

This effort was part of the thesis work of Elene Dorfmeier. The thesis, "Ocean acidification and disease: How will a changing climate impact Vibrio tubiashii growth and pathogenicity to Pacific oyster larvae?" is available online

A detailed description of her work with respect to these libaries can be found in her thesis . Page-- Raw data is available in SRA?? and a lot of secondary analysis files are available via figshare.

Communication with Elene (3/14/2014) says she has not submitted sequences to NCBI SRA.

Ostrea lurida (Olympia oyster)

Olympia oyster sequencing efforts have spawned from studies examining the influence of ocean acidification and a study examining local adaptation in Puget Sound. As of March 2014 the libraries constructed and sequencing data include the following

ID Stage Notes
Ol-larv 400_1 larvae 0112; 012159; 36SE
Ol-larv 2000_1 larvae 0112; 012159; 36SE
Ol-larv 400_2 larvae 0812; 103939; 36SE
Ol-larv 1000_2 larvae 0812; 103939; 36SE
Ol-larv 1600_2 larvae 0812; 103939; 36SE
Ol-larv 2200_2 larvae 0812; 103939; 36SE
Ol-Femalemix_106a gonad 36SE
Ol-Malemix_106a gonad 36SE
Ol-Femalemix_108a gonad 36SE
Ol-Malemix_108a gonad 36SE
Ol-Femalemix_106A gonad 72PE
Ol-Malemix_106A gonad 72PE
Ol-Femalemix_108A gonad 72PE
Ol-Malemix_108A gonad 72PE

Libraries Ol-larv 400-1 and Ol-larv 2000-1 were described as part of the publication:

Timmins-Schiffman, E. B., Friedman, C. S., Metzger, D. C., White, S. J. and Roberts, S. B. (2013), Genomic resource development for shellfish of conservation concern. Molecular Ecology Resources, 13: 295–305. doi: 10.1111/1755-0998.12052

Larvae were transferred to the University of Washington 12 h post spawning. Larvae (12 larvae/mL) were evenly distributed to six 4.5-L larval chambers. Larvae were sampled from all chambers by filtering them onto a 35 μm screen and flash freezing in liquid N2 on days one, two and three post-fertilization. Two RNA-seq libraries (Ol-larv 4001 and Ol-larv 20001) were constructed from pooled mRNA (13 μg per sample).

Raw data is available in the following locations


In late 2012, four more libraries were made and sequenced, however this time these libraries were all run in a single lane on the Illumina HiSeq.

Ostrea lurida larvae were subjected to four different pCO2 treatments (400, 1000, 1600 and 2200ppm) from a Friedman Lab ocean acidification experiment. RNA was isolated from three groups of larvae from each pCO2 treatment using TriReagent (Molecular Research Center) according to the manufacturer's protocol. RNA was resuspended in 100uL of 0.1% DEPC-treated H2O. Concentrations and quality (OD260/280 ratio) were assessed with a NanoDrop1000 (ThermoFisher). Five micrograms of total RNA from each larval group within each pCO2 treatment were pooled. Four total RNA pools, each pool representing each pCO2 treatment, were submitted for single-end Illumina sequencing at the High Throughput Genomics Unit (HTGU; University of Washington) for sequencing. All four samples were sequenced together in a single lane.

Data is available in the following locations

In order to characterize the reproductive transcriptome of the Olympia oyster four libraries were made in from pooled gonad samples. The IDs were 106AFemale, 106AMale, 108AFemale, 108AFemale.

These libraries were done in late 2012 and were sequenced on the Illumina HiSeqplatform on a 72PE run. An additional lane was run as a bonus as 36SE.

Raw data is available in the following locations