Getting ready to run some qPCRs and first we need to confirm that our RNA is actually DNA-free. Before we can do that, we need some primers to use, so I decided to semi-arbitrarily select three different gene targets from our MEGAN6 taxonomic-specific Trinity assembly from 20200122.
I used our recent differential gene expression analysis to identify those genes which were highly differentially expressed in infected vs. uninfected samples.
Overall, the process went something like this:
- Sort upregulated genes in infected group by logFC (fold change) to find Trinity transcript IDs of highly expressed genes:
awk 'NR>1' salmon.gene.counts.matrix.infected_vs_uninfected.edgeR.DE_results.P0.05_C1.infected-UP.subset \
| sort -n -k4,4
- Search for some of the highly expressed Trinity IDs in the Trinotate annotations to find SwissProt IDs:
grep "TRINITY_DN6549_c0_g1" \
20200126.cbai.trinotate_annotation_report.txt
-
Copy SwissProt ID (if available) and see what it is on the UniProtKB website.
-
If interesting (somewhat), search Trinity de novo assembly for transcript sequence.
-
Use sequence to generate primers on the Primer3 website.
RESULTS
Here are the targets and primers designed and ordered.
40s rRNA S30
PRIMER PICKING RESULTS FOR cbai_TRINITY_DN6411_c0_g2_i1
No mispriming library specified
Using 1-based sequence positions
OLIGO start len tm gc% any 3' seq
LEFT PRIMER 80 20 59.94 45.00 4.00 0.00 TGCCGGTAAGGTGAAAAATC
RIGHT PRIMER 261 20 59.97 45.00 2.00 2.00 AAATCCGCAACCAATACAGC
SEQUENCE SIZE: 334
INCLUDED REGION SIZE: 334
PRODUCT SIZE: 182, PAIR ANY COMPL: 3.00, PAIR 3' COMPL: 0.00
1 GTTTTTTCCTTTTTCGTTTTCTACATATATTAACCCCCCTTTATTAAACAATGGGTAAAG
61 TCCACGGTTCCTTGGCTCGTGCCGGTAAGGTGAAAAATCAGACCCCGAAAGTTGCCAAGA
>>>>>>>>>>>>>>>>>>>>
121 TGGAGAAGAAGAAGTCTCTCACGGGCCGCGCCAAGAAACGCATGCAGTACAACCGTCGTT
181 TCGTGAACATCGTGCGGGCAGGTGGCCCCAAGCGCGGCCCTAATTCCAACCAGAAGTAAA
241 GGCTGTATTGGTTGCGGATTTTAGGTGTTAACGATGCGCTGGACTTCCTCCTCTATATGA
<<<<<<<<<<<<<<<<<<<<
301 GTATCATGGGATGGATGCAACGAACTTGATGGAC
allantoicase
PRIMER PICKING RESULTS FOR cbai_TRINITY_DN13073_c0_g1_i1
No mispriming library specified
Using 1-based sequence positions
OLIGO start len tm gc% any 3' seq
LEFT PRIMER 65 20 60.29 50.00 4.00 0.00 CGAGTGTTTCCAAGCCTGTT
RIGHT PRIMER 215 20 60.07 50.00 4.00 0.00 GTGAATACGCCTTCCTTCCA
SEQUENCE SIZE: 237
INCLUDED REGION SIZE: 237
PRODUCT SIZE: 151, PAIR ANY COMPL: 3.00, PAIR 3' COMPL: 1.00
1 GTAGTATTCTGGAATCGGCGTTTTTTGTTTGTGTAATCCGTGGAAATGGACATATCTCAA
61 CCCGCGAGTGTTTCCAAGCCTGTTTTTACACGCTTGACCGACCTCGCGAGCGAACTGCTC
>>>>>>>>>>>>>>>>>>>>
121 GGCTCGAAGGTGCTTTTTGCCACCGATCAGTGGTTTGCCGAAGCTTCAAATTTACTCAAG
181 AGTGAAGAGCCGGTATGGAAGGAAGGCGTATTCACCGAACATGGAAAATGGATGGAC
<<<<<<<<<<<<<<<<<<<<
ubiqutin thioesterase
PRIMER PICKING RESULTS FOR cbai_TRINITY_DN6549_c0_g1_i1
No mispriming library specified
Using 1-based sequence positions
OLIGO start len tm gc% any 3' seq
LEFT PRIMER 297 20 60.00 45.00 4.00 2.00 CGGTTTGTTTGAACGGCTAT
RIGHT PRIMER 577 20 59.95 50.00 4.00 3.00 GATAAAGCTCGGCATTCTGC
SEQUENCE SIZE: 647
INCLUDED REGION SIZE: 647
PRODUCT SIZE: 281, PAIR ANY COMPL: 3.00, PAIR 3' COMPL: 0.00
1 TGCGGGAATATCTTTAAATACTATATACTCGGGTAGCGTCTTGGAATGTCATGTGAGGGA
61 AATTCAGACCCGCACCATGATTATCAGGCATCCCTGAACCAGCAAGATGCGATCCGGCAG
121 GAAGCGTCCGTCGATCACCCGTTGATGAAGAAGCGCGAGCCCGTAGGGGCATCGCTGAAC
181 GAGCAGTTCGCGGAGAATAAGAACTTCCTACAGAAGGTCGCTTCAATCGCGGCCAAGTAT
241 GAGTTCATTCGACGGGCGAGACCGGACGGCAATTGCTTTTACCGCACGTATCTGTTCGGT
>>>>
301 TTGTTTGAACGGCTATTGGGCATGTCCCGCGAGGAGCGGGACAAATTTGTCGTGTTTCTC
>>>>>>>>>>>>>>>>
361 AAGAAATCACTGGATGATGTGCTTTGCCAAGGGTATGAGCGATTTGCGGTAGAAGAAATG
421 CACGAAGATATCCTTGAAGAGTTTGAGAAACTCGCTCAGAATGACAATGCAACCGTCGGC
481 GATATCGAGACGATATTCGACGAGGAAAGGCATTACCATATTTGCTACTTGAGGTGCCTA
541 GCGTCGGCGTACCTCAAGCAGAATGCCGAGCTTTATCAATCGTTCCTCGAAGGCTATGCG
<<<<<<<<<<<<<<<<<<<<
601 ACTATAGCAGAGTTCTGCGCTCATGAAGTGGATCCTATGTGGCGCGG