Supplementary File S1. Supplementary Materials and Methods
Ribosomal Filtering Database Construction
The filtering database was constructed by first obtaining rRNA sequences from NCBI (NCBI
resource coordinators, 2013) that also had confirmed ribosomal structure in Swissprot (Uniprot
consortium, 2013). These database sequences were used for an initial filtering pass in deconSeq. The
remaining sequence reads were collapsed into a set of unique sequences and the number of times that
each unique sequence occurred in the dataset was recorded. The most abundant unique sequences
that remained were aligned against the nr database using BLAST with default settings and an e-value
threshold of 1e-6. BLAST hits that were identified as ribosomal sequence and had confirmed ribosomal
structure in Swissprot were added to the filtering database and the process was repeated until the
highest represented ribosomal sequence identified comprised less than 0.01% of the total dataset.
Verification of expression results with qPCR
To validate results of the RNAseq expression analysis, nine components were chosen for
quantitative real-time PCR (qPCR) in the same samples. To be chosen, a component must have an
associated gene annotation, and be differentially expressed between migrants and residents for both
sexes for at least one time point. A list of components used for qPCR can be found in Table S4.2. To
design primers for qPCR, open reading frames (ORFs) for each component were identified using
SeqBuilder (DNASTAR Lasergene v10, Madison, WI). The longest ORF was aligned against the nr
database using BLAST to confirm that it contained protein sequence. In the case where a component
was represented by multiple contigs, a representative contig was chosen based on best annotation evalue. ORFs were then aligned to human (annotation release 104), zebrafish (Zv9), medaka (annotation
release 100), fugu (annotation release 100), and nile tilapia (Orenil1.0) genomes from NCBI using BLASTn
to identify conserved intron-exon junctions. For each gene, two primer sets were designed so that each
product would span a different exon-exon boundary. Primers were designed using Primer3 (Rozen &
Skaletsky 1998). Primers were designed so the product would span putative intron/exon junctions, to
prevent amplification of genomic DNA. The following settings were used in Primer3, the best primer
options (as presented by Primer 3) were chosen and are listed below.
RNA from the original samples used for RNAseq was converted to cDNA using the SuperScript III
First-Strand Synthesis System (Invitrogen, Grand Island, NY) following manufacturers protocols. Each
10µl qPCR reaction contained the following reagents: 5 µl Sybr Green PCR Master Mix (2x) (Life
Technologies), 0.36 µl forward primer (10µM), 0.36 µl reverse primer (10µM), 3.28 µl H 2O, 1µl template
cDNA. qPCR was conducted on an ABI StepOnePlus Real-Time PCR machine (Applied Biososystems,
Foster City, CA) with the following thermocycler profile: 95°C for 10 min followed by 40 cycles of 95°C
for 15 s and 60°C for 1 min, and a final step at 95°C for 15 s. A melt curve analysis followed to evaluate
disassociation of product and possible primer-dimers, with a temperature profile starting at 60°C and
increasing 0.3°C until reaching 95°C for a final hold for 15 s. Beta-actin was used as a reference gene to
standardize expression across samples as it has demonstrated consistent expression between migrant
and resident rainbow trout in previous studies (Xu et al. 2011) and it was shown to have constant
expression across samples in this study. One-way ANOVA was performed using SAS v.9.2 (SAS Statistical
Institute, Cary, NC) to test for differences in expression between migrants and residents at each time
point with p<0.05. Correlation between qPCR and RNAseq expression was also conducted using the
corr.r package in R v.3.0.1 (The R Core Team 2013), with the Pearson correlation coefficient option and
significance tested at p<0.05.
Primer3 Settings:
Product size
Primer size
Primer Tm
Minimum
100
19
57
Optimum
150
20
60
Maximum
220
24
63
Primers for comp632317
OLIGO
start len tm gc% any 3' seq
LEFT PRIMER
817 20 59.80 55.00 4.00 0.00 GAGAAGCTGAACCTGGATGG
RIGHT PRIMER
971 20 60.27 55.00 2.00 2.00 GAGAGCGTGAGGTTGGTGAT
Primers for comp650365
OLIGO
start len tm gc% any 3' seq
LEFT PRIMER
1159 20 59.87 50.00 6.00 1.00 CACGTCTTCAGGAAACGACA
RIGHT PRIMER 1289 20 59.45 60.00 5.00 3.00 GAGGGCCTGGCTGAGTAGTA
Primers for comp653818
OLIGO
start len tm gc% any 3' seq
LEFT PRIMER
2152 20 60.09 45.00 6.00 2.00 TTCATGAGGAACCACAACGA
RIGHT PRIMER 2300 20 59.75 55.00 2.00 0.00 GAGGTGTGTGCGTGGTAGAA
Primers for comp550478
OLIGO
start len tm gc% any 3' seq
LEFT PRIMER
516 20 59.87 50.00 4.00 2.00 AGACATCTGCCCAAAGTGCT
RIGHT PRIMER
633 20 60.40 60.00 5.00 2.00 CCCTCTCCTGCCAGAGTACA
Primers for comp604956
OLIGO
start len tm gc% any 3' seq
LEFT PRIMER
1239 20 59.84 55.00 3.00 0.00 GAGGGTGCTGAAGGTCAAAG
RIGHT PRIMER 1388 19 59.80 57.89 2.00 0.00 ACACAAGGGTCAGGGGAAC
Primers for comp630440
OLIGO
start len tm gc% any 3' seq
LEFT PRIMER
582 20 60.39 60.00 3.00 0.00 GTGTGGGGCAGAGACATACC
RIGHT PRIMER
742 20 60.67 55.00 6.00 2.00 GTTCCTGTATGCGCAGTTCC
Primers for comp642622
OLIGO
start len tm gc% any 3' seq
LEFT PRIMER
1030 19 59.81 57.89 6.00 3.00 GTGAAGCAGGACCCTGACA
RIGHT PRIMER 1178 20 60.12 55.00 4.00 0.00 AGGACCACTTCACTGGCATC
Primers for comp642886
OLIGO
start len tm gc% any 3' seq
LEFT PRIMER
236 20 59.84 50.00 5.00 1.00 TGGTTCAGGTTCAAGCCTCT
RIGHT PRIMER
381 20 61.17 55.00 2.00 2.00 GGGGATGGAGCCGTTAGATA
Primers for comp652313
OLIGO
start len tm gc% any 3' seq
LEFT PRIMER
1123 20 59.84 55.00 4.00 2.00 GAAGTGAAGCCAGGGTCTTG
RIGHT PRIMER 1282 20 60.07 50.00 7.00 0.00 ACAGGATGGCATCTTTGGAG