Primer Design - C.bairdi Primers for Checking RNA for Residual gDNA

2020
Tanner Crab RNAseq
Author

Sam White

Published

February 20, 2020

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:

  1. 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
  1. 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

  1. Copy SwissProt ID (if available) and see what it is on the UniProtKB website.

  2. If interesting (somewhat), search Trinity de novo assembly for transcript sequence.

  3. 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