Tuesday, May 19, 2015

5 18 2015 Target List to Usable Primers

Last week with the guidance and assistance from Steven, we turned the Oly Transcriptome V3 into annotated list of possible genes for qPCR investigation. You can see Steven's notebook about how he discerned which genes would be useful for our study. The list which contains sequence ID's that relate to the Oly transcriptome V3 Fasta file.

Being a fool I decided to try to hand search the fasta using wordpad and the list of ID's. Each ID took approximately 5 minutes or more to find in the document. The fasta was so large that it was nearly impossible to scroll through because it lagged my computer. So I decided to find a coding way to search the file quickly.

First I attempted to find a way to do it with python as it seems like a quick way to write a few lines of code and produce a good output document. The samtools package contains a function called faidx which via extensive research seemed like it would do pretty much what I wanted by search the fasta using the input sequence IDs and produce an output document with the related sequences. Sadly because I'm on a windows machine, samtools is a nightmare to install and I couldn't find a good work around. Normally I would then port everything over to eagle and run it all remotely which is pretty cool and easy to do. Instead though, Ryan Waples suggested I look into an R package called SeqinR (Seq in R, get it!).

I'm a much better R programmer than I am at python or perl. The other nice thing about R is that it works on all OS without needing language specific dependencies. So I popped open R, installed the SeqinR package and began playing with my data. At the bottom is what I did in R to search the fasta using the unique sequence IDs.

There isn't a good way that I've found to produce an output document from the list function in R. Instead of figuring it out I just cheated a little and copy/pasted from the command line output to a google doc.

I then reviewed this video by Steven on how to make primers




Following his example and doing some reading on proper primer design. I took each sequence into NCBI Primer creator to develop primers for each sequence. I copied the sequence into the input field and select a product between 100 and 400 bp and disabled the specificity check for the primers. These options are highlighted in the image below in yellow. 

After hitting "get primers" I was lead to the next page with a list of possible primer combinations. From these combinations I need to pic primers that would work well for qPCR. From my reading on qPCR primers I chose to primers based on the lowest Self Complementarity and 3' Self Complementarity values to reduce the possibility for primer dimers. You can see an example of this in the image below. 


Once I selected these values I then copied the FWD and REV primer sequence and product size into a google sheet to create a list of primers to order. You can view this list here. From this list I'll order primers and then check them via qPCR to determine if they produce interesting results. Hopefully we'll see some cool differences between populations using these primers.
seqinrnotebook.R
##install.packages('seqinr')
require('seqinr')
## Loading required package: seqinr
## Loading required package: ade4
olyv3<-read.fasta(file='C:/Users/Jake H/Documents/FASTA/Olurida_transcriptome_v3.fasta', seqtype="DNA",as.string=T,
                  set.attributes=F)
targetlist<-read.csv("C:/Users/Jake H/Documents/GitHub/Resilience-Project/short-list-olyo-v3.csv", header=F)
olyv3[c(which(names(olyv3) %in% targetlist$V1))]
## $comp6939_c0_seq1
## [1] "tggcgctggaaaatcgatagtcaagagcctgtacttcagtatttactttggacggggttaactagtccctagtgaccatatcgagagtgaatatgtattcaatggcaggcatacaacacggtagaagattcacgtcggattagcagatatatcattaaggacaattgttaccgccctcactgattacccacctttgttttgtgagcaaacatttattaagcaacacaccgccctacgccagaaacaaccaagattctatcatctatacaaaatcaatgcaccctgcgcatgtcacaaaacgttgccatgattttataatcatgcgacattaggaggcatggctctccacatgtaaactctggatcggggtaaaaaaaatctttttttaaaaagcaggaaactatgagtcttgtgtgcggattttccggacatattgaccgaaatgaaacgaatacctcaaattgctcaacgaattgctctactccaaattgggtgggcacggctctcatgagctggattagtacgaaaaatgtagttaattctggagtgatgtttgcctgtggattttttgggaatttgttagctttaattgtgttaataaggtctagtaaggaacagcgacggacgattttctaccgtttggttgctggactgaccatcacagatttaattggcaccaccttgacctctcctgtcgtgataacagtgtacgtaaagagacgctggatagggggcgacttcctgtgttcttactttggatttatgatgatcatggcgtgtttctccacgatgacaattgtatgtgctatgtctatagaacggatattgtgcataagacacccatatatctattatacccgattatcgaagaagcacgccaccattattttagcatcgtgttgggtaacgtctgccattatagcttcgttgcctcttttaggatttggggaaattgtcagaaaatacccaggaacgtggtgttattttgattattactcaaaagagccgatccatatagtatttaactatttcatcagtatcttagccatgagtattatcattgtgaccatttgctgtaatttgacagtcatttataccattctaaaaagcagacggaaacagggaatactccggaaagaggacggtacttcacgctccatccatggtgccagcaagcgttacgcagaatgtcagatgctgattcttttggtagggatcaccatcgttttcacctcctgctacactccgctatacgttcgagttatcatgaatcaaacaggcctcgtttgcaataccctcttggacttacaactgatccggctagcctcttataatcaaattctggatccttgggtttatatactcctccggagagaactcttgtggaagataatatctggcgtgaagtacatttgtaatcgaggtcagacagacctaaattcattaccccagaaacaatttgacgtagacaatgccaactgctgcgtgttttgtttccactgtttgtgcgaccctccggtgactgctagacagcgatgtgggtcaattttctctcaggacggccaatacatacatgcatgtccaggaagaagcacaatgaacgcctctagtttttcaaacctcaaaagcacctctctgacttctttgaacggaaggcaatcgggccctctgcgagtttgctctgcgaatgacgtccatatgctactgcttaataacacgctgaagaaacatggagtgacaaagagtacaagcacacgttttgtggatgacattgagaacgactgattcattcacaaatatttttacacgtgtttcatttccaatcaagtgatgtacaatcaatatttaaggattttatatacatttttactacttattagccttacaatttttcatactgtatgtgttatcatgtttatctaacattccacaatcttgaatgcgagatcatatattttaacactgatgaggcaaaacaacgtacatgagattactagaggattcccatttttattggcttgctttacttttgtggcaaccagtacatgtaccagtggattttttcaaattttgttgttgtgtaaaaaatcaacatattttccttactatcaaaactggca"
## 
## $comp7183_c0_seq1
## [1] "aaattgacattaagggattatacgtgcaacgagtttttcattaataagtaaacgattatctcttttgaccggaacatataatgaagttttcaagaccaagtatttaaagtgttgttattttcaccagaaagtttaaaggaatcggagtgcagttagacgaggacagcgatactatataagctggaacttattaatacacacgtactctcgtctcagtataaaccacctgttccaccataaacatgatttattaatacggttatttaaaccttatccggttgagaatgtgtgtgcaatcagagctattttcgttgaggagcaaatttagaatcgggatttactgactgaacaagcggttgggaaaacaatctcttgcacacaaggatcatttactttcagtctatttggaatttatagaagaatgtgctatgacattaaataggggaattaacaaactaatggaagaccgtgcaggaaccaagctctatattcgtgcattttcacaatgagtttctgtcgatttggttttctactgttgctagcgtgcaccgggctggctggatcgtcatcgccttctcttctagaatcggcttttccttctcactacgacgaaaatcgaagaagagaaataatacaggcttttgagaaaaccttcttgcatctgtttggattgaaggaacgaccaaagccaaagaaagacacttttattccacaatatttgttagagttatatgaaaaacatagcgaggacccagattctcttggagatagcgtcaatgtcaaaagacgaggcgcgggttccgccaacactgttagaagttttcttcataaagaattgaccactgacgtcattagtatggcaggatgcggggagcacaactgtgcccatttctacttcaacgtatcatccattcctatggaggaatcattaactggtgctgagctaagactctttattgatcaaaataacgacacaaaaaatccagtcggaaacaggaaacagtttagacataaaatagaaattcacgaggttcttcaaccggaaacccaacattctgaagccataactaggctgctggatgtgcgtcacgtgggaggaagaaatgcatcttgggaatcttttgacattcacccagcagtattaaaatggaaaaagaatcctcatttaaaccatggattaaaagttcgtgtattacaatttaaaaacaaaccgtccacagactccattaaacatgtacgtttacgacgtgacgtagatagtgtggaggaagagaggcacacgagacctctgttggtgactttcacggacgataaccgtgggtcaaggactaaaagatccaccagtgataaaacaaaaaaaggtagaaaaaataggaaaaatagaaaaaataaaaagcgaaggagaaatagaaagaaaaacaatagaaagaataaaacaaagaggaaaaagtataacgaccagtgtcgcaggaaggagttaaatgtagattttaaagccgttgggtggaacgattggatattcgcgccacccggctataatgcgtattactgtgatggttcatgtcactggccttatgatgaccatatgaatgtgacaaaccatgcgatagtccaagacttagtgaactctatagaccctagggcggccccgaagccctgttgtgtacccacagaactcagttcgctttccttattatatactgacgaacatggcatagttgtgctgaaggtctatcaagacatggttgtagaaggatgcgggtgccggtagccatgataactctttaaattgttattgttatttgttgtgatattatttatttgcgaaaatggatgaaaaagtgtgtgtgttccgaaatatggctgttcggaagttagcttcatatttgctcatttccaatctcccattcattgacaatcatatcattattttcattttgcataaattgcat"
## 
## $comp7220_c0_seq2
## [1] "gagaaaatggcgtcaacttttgacaacgtgcaagtcgccgttctcacagaaaagtacaatttcaagtttctcacagatgatccgctcattcttgatatatcacaccaaggagactgtgttatgctccagtttcacaaagacaaagttcctgtacacaccttgtcagtaaacaacgacacagaacatgccaggatgggatcacagagtatcatcttcacacgagacaaggacacaacactggtccattttcctagtgttcacaatatgaaacagtttcagtttcgactgaatgaactgaaagatggattaaaatctgtcttcagtgatagaacagatgcagcctcagcagttcaatatttccaattttatggatacctctcacagcaacagaacatgatgcaagactatatcagaacgtccacgtaccagagggcaatgctggcaaacttaatagatttccatgacaaggttgtgttagatgttggtgctggatcaggaattctgtcgttttttgccgtccaggctggagcaagaaaagtgtacgccatagaggccagcagtatggctacacactgtcagacactggttgcacagaataaattgtccgacaaaattgttgtgatccctggtaaggtggaagatgtggagatccctgagccggtggacaccattatatcagaacccatggggtacatgctgtttaacgagagaatgctggaaagctaccttcacgccaaaaaatggctgaagcccagaggaaagatgtttccttctcagggagatttacatatagctccgttcacagatgaagccctctatctggagcagttctctaaggctaatttttggtatcagcaatcattccatggaattgatttgtctagtttacgcagtgcagctgtcaaagaatacttcacacagcccattgtggatactttcgatgtaaggatatgtctggcaaaatctgtcaaacacaccgtggacttccagacggcggaggagactgacctacacaacattgagatccccctcacgttcagcatgcacgagtcggggctcgttcacggtctcgccttctggtttgatgtggcctttttgggatcagtagagcctgtttggttatcaacagccccgacagagactctgacacattggtatcaggttaggtgtctggtggaaacaccggtcctcgtcaaacaaggacagactctcaatggaaaatgtgtgctcaggtgcaacaaaagacagagttatgatgttgatattgaactgaacatcccaggaacaacctcaaagtccacaaacacactggatcttaaaaatccattttttcgttacacgggtcagacaccacctgctcctcctggggtcaacaactcttcccccacggaaacctactggaataatctggcgacaggccagactgtatacatgaatggactggttaatggggacaccaatgctgcacagcttctaagccaccagggagttattcaacaaaatctcatcccagtctgtacgttgatttcaggtttatctgacccatcccaaagactttattgaatactttacagaacaacagatttttacattacagaacaacagatttttatctgtttatttaataatgaaatacttgtacaatgtatatatgtgtatgaaaattttctatcaaataaag"
## 
## $comp8243_c0_seq1
## [1] "atcaaccaaagacgaatttatagactaacttcaacgaaattaccaaattgttttccattgtccatttacagaacagataatacatatcaaactgaactatagctaggatacaatatacggcattaagttacaatcatcataaaaatgagaaaccacgcagggagaatggtggtcagcctaattctaacattggcgctgctaactgccgccgcatcaacgtcatcttccgacgatagggcggatcccgtgataggaatagacctgggaaccacatattcttgtgttggaatattcaaagacggccatgttgaaatccttccgaatgagcaaggaaaccgaataacgccatcttacgttgccttcgatgctgatggcgacagacttataggagacgccgctaaaaatcaactgacgtcaaaccccaaaaatacaatctttgacgttaaacgattcattggacgagaatgggacgatccaacagtacagaaagatgtgcaatattatccgttcagcctcttgaagaaacacaacaagccctacatccgagtacttgtaggcgaacaagaaagagtgtttgcaccagaagaaatttccgccatggtgctggggaaaatgaaggaaatagccgaggggtatctgaagcaaaacgtcacgcgtgctgtaatcaccgtcccggcgtattttaacgacgctcaacgtcaggcaacgaaggatgctggtacaatagcagggttagaggtgctccgaatcatcaatgaaccaaccgcggctgccatcgcttatggtctaaacaaaaacggaggagaaaagaccgtcttggtatttgacttgggaggaggtacgttcgatgtgtcgcttttgacaattgaccaaggagtgttcgaagttgttgcaaccaatggcaacaaccatcttggaggagaagacttcgaccagagagtaatggatttcctgatcaagaaacacaagaaatctacaaatatggatttacgaaaggacgcccgtgccctacagaagttacgccgagaggtagaaaaggccaagcgaacactctcctatcgtcacgaagcacgtattgaaatagagtctctaatgaatggcgaagacttcttttacactctcacgagagccaagtttgaggaactgaacatggatttattccgatcaactttaaatcccgttaaacaagttctgaaggacggtgatatgaaagcctccgatgttgatgaaatcatactcgtgggcggatccacacgtattcccaaaatacagcaacttgtcaaggaattcttcggcggaaaggagcccagtcgtggtgttaacccagacgaatcggtggcctatggtgctgctgttcagggggcagttctaagtggagcagatgaaactggtgacgtgcttgttgtggacgttaaccctctcaccctgggaatagagacagttggtggcgtcatgactaaactgattccacgtaactcccccattcctaaccggaagtcacaaatattttctacagcagccgacaaccagccaacagtgaccattcaggtattcgaaggtgaacgacccatgacaaaacataaccaccttttggggacattcgagttgactgggataccacctgctccgagaggagttcctcaaattgaagtcacttttgaaatagatgcaaacagtattttgacagtatcagcagaagacaaggggaccggaagtaagaataagattacaattcaaaatgacaacaatagaatatctccggatgaaatagaaaaaatgattaaagacgctgaaaagtttggcgacgaagacaaaaaagccaaagaaatcattgacgctaagaatgaactagagcattttgcatattcgcttaaaaaccagttaagtgataaagggcaactcaaagataaactcggtgatgaagataaaactacaatccgagaggcagtggagtctacgattggctggattgagtccaatcctgacgcggaacttgatgatctcaaggcaaagaaggccgagttagagaatatagtacaaccaatcacgagcaaactttaccaaggaaatccaagttcaaaggccgacgaccgagaggggctttaatgaaaggctttcctttatacaatcggctgggccctgatctgtgatattgataagaaaatgtcccgtcactatttattgccatatctcatttatatcattccgtgcaatgctgtacattgttgattttattgtttgttcagtattttttatattaataaataaaacggtgaa"
## 
## $comp9280_c0_seq1
## [1] "ctccgggctccaatcggaactcctcggaatgctcgtggcgagttccgatcgcttaggcctacgcgtacgtttgcatgggcatgttttcaaaaatttcaaaaatttccacagtaaaagtattagtcaaaataatttcttatcaaaaatgagcaggtgctaaagctttcttagcgaaaacttttattagatcgaagagggaagtatatttatgtaggtcagttggccttcctaccactctttgaaccagccttaccaggattaaaatgtcagatctaaatagttccgtgagatcaaaatgcagaaagctcttcgtagcagccatcgattttggaactacttactctggttatgcattttctgccacatctgattggtccaaagttttgaccagtaactggacaggagggaaggttgtcactctgaagacgccgacagctctgctgctggattcagacaaggagttcatggcatttggatatgaggccgaggatcagtatttagacttggctcgagatgacgaacacgaggattattatttctttcatcgatttaaaatgattcttcattcagaaaaagcaccaatcagtcgaaaaacaaagctaaaggatgccacaggtaaagaaattgaagctataaaagttttcaaactctcaataaaatatcttgtgggggacttgatgaaaactttgagaaacagctacccggatttaaaggactcagatgtggagtatattatcactgttccggctatttgggacgatcgatcaaagcagttcatgagggaagctgctcgagaggctgggattgacaaatcttccttgaccattgccttagaaccagaggctgcatccattcattgtcagtatttaccagccaaaaacctcaacctgtccacttccaagtaccttggggagcttgctgttggggacaaatatttagttgccgatttaggaggtggcaccgcagatataacagtgcatcacaaagttgatgacggatctttggaggaaatgatggaagcaagcggtggaccttggggaggaaagtctgtggatgacgaatttgagaattatattcaggaaattattggagaaaaatccatgaaggagttaaagaaatcctgtttcgatgattacttggaactaatgaggaacttcgaattcaagaagagatccgctaagcctggaaaagaaggcaatacacatctgtctgttccactgggtcttgtgcaaattgttaagaaagacaaatcacgaaaatctatggagaatgccattgaaaattctccacatgcaggaaaagtcactttcgagaactggaagttgaaatggaaaaatgaagatttccttcggttctttgaaagcacaatcaagaaaattaaagaccatatacggacaatattgttcgatagtgatatgggaagtgtagaaactattctaatggttgggggttttgccgaatgcgaagtggtccaaaagtcggtgagagagtctttcaagatgaaacgcgttgttgttccagaagatgctggtatttctgtattaaaaggcgccgtatattttggacacattccagatgcaatatctagacgaacagctcgatatacgtatggtatccaaagctggcccgcattcaacgcaaccaaacatcccgaaaacaagaaagttgaagtgaacggtaaagctagatgtaaagatgttttcttcaaatacataactaagggacaacatttaactgttggatatcagaaatcacagatttttcaagccttaaacccgaaagagaaaaaactggagtgtgcaatatacatctccgacagcaaagatccccaattcgtagacgatcctggatgtcatcgacttggcgttttggttatccccttgccggatctaccagatggcgcatcgatcgaattagaggaaagtatgatttttggcgaaacagaattgcaacttcaagctaaagatatatactctggaaagtcgtacgaagtacagttagatcttttagcagaaccttcagaggagaccgattagagatacagtattcaatatgaatttacacaatgttggagatttacgtgtctaacttagaattggcgagtttttgacatactaaaggcagtaaagactaataaatgttagctcattagaccaaaatcgttcaaactaaatatgtattcatagtatgcatgttctacctactattagaatacattgattgaatgtgctctatgtgaaataaatgtattaaatgttaaaacgtactgtttgtagtttgtctttcccatctttactagacgtattgttatagaaaattatttaaatgacgatgcatacaagaaaagttgcacattgtttattaataaagtggggtcttggtggagtggaggatacattgacttcacaattgttagaaaaatctttagagatgttataatccctaccctcaaaattgtaccttggaaaggggaggagaaggctcatattcctttactacataggtttaattcctctgttcaagcttaaatttgtaccattcaattctaatatg"
## 
## $comp9524_c1_seq1
## [1] "aacatactaagttaaattcagctcctacaaatgaagttgaaataaaaattttcagctataaccaaatagtagaaacattaatagaatccaggacaatagatgtgagtagagatggctgggaaatatttgacattacccaagtcgtcagagactgggtcgataacccagaattaaataatggagtagaaatttatgcaggcggtctcaatgccggtcaattactttttccctccgtggatgtcgcagaaagaaagagctcaaaatataatagaacagcaaccagacacaatgttgtggtaccaattctggaaatgaagactcatgaacgttcgatattaaaacgtgtcaaacgacaaaataacattgagaggagagattgtgtcaaaggggacggagaaagcagatgctgtcgctttacaacgacaattgacttcgctgatttgggctgggacgactggatactcgcaccacaacattatgaagcgcattattgcgatggaagttgtcccgatcgctttaaaatggccaacacgttcgcgggaatacaggccagacttcacgctttatatcctagcaaatttccgaaaccgtgttgcgttccatcaaaacttagcccacttacaattcttcataaagacagcgatggaaaatatcagttcacggactatcccgacatggtcgtcgaggactgcaagtgcgcatgacaggaaccttatgcttatttgtgaattatccttttgtgttcaataattaaagccaaaaacgttcccgactttgtgtgtcaacattgctttgactcaacttcatgctcgcacaaagggtaatgaatgtaataactttcaaaatgctactgttaaaaatcgcgcaagtcaacaatctactcaatgccttttttactttgttatttctatactctgctgttgagttccaagatggctgtgttaatttcgttagagggacaatttgcaagcccagtaaaaagcaacgatccttttcgtaggtgatattttacacaagtcaattactatgtggttttcgcatatttttcgcaaatgtaattttttttatgtgttcaaacgtacaagagtctatatctttactcgtaaagagcaatgaaagtgcatatggaccaattttcggagaccgaagagaatctcatgtttgtgcacgatatctgtgatcttgatatgcgtgtatttatatgtgtgaatgttatatgaatgctatatgtatgttcagt"
## 
## $comp9638_c0_seq1
## [1] "ttccataagcctcctattttattgtcaaataaatatgtttcctttccaaaatgaacaaacatgatgtagaaataatcataagggccacatcctttcactaacaattgacatcatattgacactttatttcaactttaaataccagtaaatgtatgtacacaaaacagatttctgacagaaatgaagaaagacggtatcttaaggtgtagaaatttgttcatccatgaacagcatttccagtgataccaagtctgtgcacagatacaaagtggtcataaccgctttcaatgatgactctcatgtgagtggctcttttattgtttgctgggaattcctcttgctgaagagaattgtctgtggaatcgagctctttttccaacaagtcttcaaaatttgttggatcagtttctttactggtttcaaatgatattttctttacattacaacacgctatttcaatacgctgtatactcatcatagaaggaaaggtgatgatgaattcttgtgggaaaagaccagtagatgcccaaaatgtttcctggtctccgtcaatgatgttttcaggtggatgattcccatcactagaagtcgctaagacaacctgcgctccagcacttttcagagcaacatcaaacatttcaatttgcaacaagttcccctggcgttgatggcaacaaaatatttcatcaatgttatttggg"
## 
## $comp10127_c0_seq1
## [1] "aaaataggaactacacaacaacgcggaggtaaatatcagggtcaagatggaggcgatagcgaaacatgatttcagagcaactgctgatgatgaattaagcttttccaaaactaacattttaaagatcttgagcatggatgatgatcagaactggttcaaagctgagttagatggtaaagagggatacgtaccaaataattatatagaaatgaaaccacacccttggtttgctggaaagattcctcgattagaagcagaaagacgattattggagaaagactgtcgagggaactttgtccacccagacggggcattcctgctcaggaatagtgagagtgccccgggggaattctcaatatcagtaaagtttcaagaaaatatccagcattttaaagtattaagggatggagcagggaaatatttcctatgggttgtcaagttcaactctataaacgaactgatcgaatatcatcggatgtcatctgtcaatcgtggacagaccattgtactcaaagatatggcgactaacaggggaaagaaagcgtcggcaatgtacgaattcaaaccagaaaccgttggggagctagcactcagcagaggtgatatcataacagttctagaggaagtggactgcaactggtggaaaggagaacgcaacggcgaggaggggttgtttcctgctgcatatgtcaaaaagcttgattgacctgcaattgtgtcgcgaatcttgcttgtggaaaattagcttgaaaaaaaaatcgtttgtaatgtcatctttgatcgggacaagcttctcatcctgtctattctgatggattggtccagtatgtcagcgcagtgtttgatatatattggcactcacgaaaaaactactatttgttagccatagtgtgccagccttattacgtatacccatcttctaactgtgtaccacgggagaccttccgtcagtagtctcagaaaacgattctctttccaatcaactagttataaagattttttttttccaaaaagatcaaagatgtacacatgtatttgtataaatttcattttcatatatagatactcattgtacataactgaacattttgcagaagtatatatgtggcttaattcatatttttaagcatttctgtgcaagataataagaaagctcttcatttgaatagaagtagaataggaccataaggtacaggagagaaaacataacaatccaaaagaatcgtgtcctcaaatgtgagaacttgcaaattgcttggagattcaacaagaaaccatagacaccattaggagctgttggacgtaagcgcagtaacacttcagacactgttgaagctgcaagtagatctgatgacatgagaattatagatttgtgtgtataaccattttgaatttctatatcctggttctagaataatatacattctgttggaagttccatatactaattttcagttatggttatattgatgcttgaatggtctccttattcactaaatatcaaatgttctgcgtatatttttttaatgtgtacaaattttagatgtatgttttttgaagggattcttgcataatgatttgtactcagagactgaaatctgtatggaccggtttgcggtttagtaatttccaggcatagattacagaggctaatgatgagttattgattgaaatcgcaggttattgacatgtttatgttgatgttgatgtatggatatatatatttatatactataactacaatttcttgtgatcttgtttttgtactttcactttcattcttaaaaacaattgcatgactatcttcttggcatcaaacaagttttgaaaagacaaccgctttcacattctttctgcagtttgtgtggcaattttgtctgttaagctaatatgatgctttaaatttctgcgttcttcctctaaacacatatacttccaatgtcattatgctggtcaagacatttttgcacttaatagtaatataacttaacagtggcactggtggtatatgagttagtttctgagaaacagtgtgtcctctgccatattttatgaaatgttaacaagattgtcaatatgtcatgaataaatttagcaatttttaaaaaaaaaaaaaaa"
## 
## $comp10930_c0_seq1
## [1] "gaacagagactgcaaaagacaagaaacaggccgcagcagccctgctccctctagcggagtttgttggggccaccgtgtcagcgccgttatttttggccctagtggcagcctacgggctatcagctgttctggctcaccacattacacaatcctcctccgacagccattcctgtgctaataatcggggctggtgccgatcgcaatgtttctctcacgaatacgttgatcactaccacagtattgtttgtgggttttacgtatgctgtagaccaggcaa"
## 
## $comp16251_c0_seq1
## [1] "agcagcaatcaattcagaaacaaacattcattagcaaatgtactgcagcagcaatcaaccacggaaagaaacatggacactggtttctaataaaaacaccaatggtaaaggtcttgattaaaaccgaaacaagtgtttgtggcatgaaaccaagggcgaaggcagtgctagctccaaaacacgtgaccactattttactgtaaaaagtaacaccggttcttgttaataaatacatcaacaatgttctatgtggaaagagagtattgcatctcaatcaacaaattctaagctccaaacgggtttgatagatttgtgttttggaactcttcttataaaaaaaaataaataaataaataacttgcagtttttgtcttaccaaaatcacttaacataatttacagagagatgcaaacgtttcacattaaggactcgtgggaaatggtttgttacatataataattactaacatacatgaacaaaataacaatgataaacatactattaatttagcagggccttatgtaagcgactaaatgaccggggcatcacttctcacgttcggtatatgacatcataatacgtttcacaaacttacaactaccatctatagttggtcaaatgaattaagaaggtctaacacatctccatattataacgttgacctcgttttgaaacactgaatgaaaatatgcatatttgcaatacttatataatccttaaaaatataattacacgtgtacttagccgagtgcgtaaccgagcccactactggtgtgtaaatctacaattcggatctaacagggcttctattcccaaactttctttcctccaaaattgtgtttttccactaaatggtaaacaagacagttttcaagtttaaaatagcattgtgcatatccttcatttctcaatccacagaagatttttcaggtgtgtcatccctccttaaaatacaggaccgcacgaaatgaaaatctcccatacagtgaataaaaaaatcgaccctgggctggtacggtatggaaggtagcatgtaaccatagtacaataacaaccaatatacaatatggaacagtaaaatgtttacaacaacgataacaattcattctctatggacctaacggggggagggggggggggcattgatcaacaacacaaaagacccgtaattcttcgtacacactcgggagatttctattttgtgcatggctacagtgagttacctacaatcctggatacattagcactaaccattagacactaccagctgcttgcactaacgtagacagcgaagttatacttcgtcaggatacagcgaaacaaatatgactggcaaaggtacaaaagaattccccactgttaccagtgctattcggctactaggatatatgttcatgttattatggcttctttcacaaatccaaatgctatggtacactttcccaatggcaattacctatggcaacagtagaccacggagaaccaaggtttcctactataacatcaaccattgaattactggggagacaatatattgtttaaaaacccttggatttcacgtatcggtatcaatactacaaattaaaatcttcagttatatacagcacctgtgaagtttaaaacactatgtcgaggtatatataaatatttttccatgtgaatcgtttcagctataatatgactgtccctcattaaacaaatccaatagctgcggaggaatatcaccttggtgatctagtcggatgcacaacacgcgctctgcatgacgggtactaatagtgcgcagatccgtcactttcatcagtatcttagcccagtgcactggttggtgtgggcgtttggtcgctatgtaacgtttaaatgctcccagaatggaatcctgaagtttttccacagcttccggttcctttaaacctgaacgatccgactgcatgagcaacacagaagcaagtaatgcgagctcagttttatccagttttagtttagaaaggccaacagcaaattcaaatattggttcaacaaggagacctaaaccttctgatttgagttgtgatttgtgtagacagacgccattatgcatggttatggtctctttttccggatcgtaccgacaggcggcacgcaaacacatgatctccatgcagcacgactttaatagcaggatctggtcgtccacgaacaattgaataaatcctggtacatttttggcaaattctacaactttgacaatagagggcgtcatggcttccgctaactgttgccaagcttcaggatctgaggggggtaagccatcttgggtagcagactctttcattggcactttgacaacagtttgttcataagctgacgtaacttccgctataatcgtcttgtctgtttctgtcagggtgtcatggttacacccttctgtcggctttggttttactttcatagtctcttgttttcttttttctctattgtcttctatcagtttccgtttggcttttctttgtttttcattcaaaacaagatccgttgccatgccaacgtttaagcattttctaaatctacactcttggcactggttacgagagtttttatcgatcgaacacattttattccacttgcagtgatattgaagattcttctgtattgtgcgacgaaagaagcccttgcagccttcacaggtcatacatcgataatggtatcctgtggccgcgtcaccacaaacaacacacggctctggtccattagtgaggtccatgtagctgggaatgtagggagtccctttcctcttgttggccgccgcaagatcccctccccccgcgtctgacgaagcctgtgacgatgaaggcgatggcgaggagttaaaagcactgatgggagaaggcatggatgagttgtccggactggacatggaaacagaggacagtccagcatacgatttggccccagcttcacagacactatcctcactcggcgatggtctctgtggcaacactgcataagatggagtaggtcctgttggcatcatggcagattgaaagtatggcaaaaatcctcgattcattccgtccacaaattcattctcctcactacctaactgaatgtccattgtgaaaaactaaatcgttcaaggtaaatgtgtagctgtttacactcacgaatgatatcactacctatccacacaatccgaaactgaccaactcaatgtccgttctcatcgatcgagagtttacgccattaataaacacacatgtaacattgttcattcgggttgaatttcaagggggtggtggtggggaatattctatatccttgtttactcggcaccccacatttgtgcgtctacgacatggtcaacaactatagaatgtgactagccgatgtgatgtaacgccccagcagttcaacgctggtcggatccactatagagcgaaggggaaatagcatttttcctgacctgatcattcctcgactttctgtcctgattccatgcaccatacagcctggtcactgttat"
## 
## $comp16752_c2_seq1
## [1] "gaatatcaaaacgtttcgctctttctttgtctcaccggctttgtgcttgtctaacccataagcaattgcagcggctgttggctcgttgattattctgagcacgttgatgcctgcaactttggcggcgtctttggtctcatgtctctgggagtcgttgaaatatgcagggactgtgataacagctcctcctattttctgaccgaggtagtcctctgctgtct"
## 
## $comp19002_c0_seq1
## [1] "atttagacagttgcgtacctttgatcttgaggatcccgccaacaccgaggaggacacgaaactccgcccacagctggaactttgtgttgcgaaacacatggcaatttggcttggtatcacccttggtttcaatgggatcattctgctgtttggtatatttctaaccttcgaaacaagaaaagtgaaattagaaaattataacgactcccgattcgttggaatggcaatctacaatattacggtgcttggttccattacagtaccattatgcctcatcatcagcaagcaacatgtagcaaactttatctttgtggcaggttgcatccttctctcttgctatttgtcaatgggtcttctgtttcttccaaagatattacatgtattgcggaatccagaacctgtccgagatgaaagatttctagatgatttcgggaccccaaggggagaggtggacactatccatcagagacttatacgtgaaaatgaagatttgaaaagacgtatacagatgatggagaacagagtggcattgttgaataaacaactgataaataaatcgtataaaagagtttatctatcagctggtcatcaggagtgtggtgcacataaaagcagctgattgacttaattaattcggcgtttca"
## 
## $comp19571_c0_seq1
## [1] "tttttttttttattaagagactacatttattgttaaaataagatacttgcacaaatctaaaatattacaaaaagacaacacaatgggggacacctgtataaaaaaaacttttgtgaaagtcaagagtttcaagatttgaacagatagaaataagacagtccagaacaagaatacataaaaattgacatatttccctgactattcatgatagaatgaccagcatggttcaatgcaagaaatcaaaagtgttaatcaagaaaaccatggaattaactttctcaatcacatttctatgcatgcatataatggacattatcaaaaccaacgactgttgatctgcattaaataagaggtgtcttacagaacataaattaaaaaaaatccaatgacaaatgagccacacaatgagaatttgtctggaatggaaactaaattaccatgccatgtcctgaaaatgtctgctgcattcacgataaaaatttcaatgtctatatgaaagtaagtttgtgtgatgaaacagaaacgagatcatgcacgctctcctcgaatcctcctagccaactgaatgtctttgggcatgattgtgactctctttgcatggatggcgcacaagttagtgtcctcaaataatccaaccaggtaagcttcactggcctcttgcaaggctccaatagctgcactttggaatctcaagtcagtcttgaagtcttgagcaatttctctcaccagacgctggaaaggcaacttcctgatgagcaactcggttgatttctggtacctacggatttctctgagggcgactgttcctggcctgtatctatgaggtttcttcactcctccagtggacggggcactcttacgtgcagcctttgtggccagctgtttacgaggggctttacctcctgtacttttacgtgcagtttgctttgtacgagccattctgtcaaatttacttcttacaccgactaaccgatccgacgtttgtgaatattttaactcttgtcaagtttgtatgttatatt"
## 
## $comp20292_c2_seq1
## [1] "tggagatgatatggagcgagaagttttggtttcccgagggcgtgacatgggcagagttacagaatgtgaacccaaaggtgtacctcccacagattgaagatatcggatttttatccatatttgtcggtctcctcctgctaatcacacgatttctgacggagaggttgttagtgatacctttggctaagtgcttgggagttaaagttagaaaacatctgcatcttgcaaaagttccggagttagagcagctttacaagtcaaagaaaactgatgtcaaatctatcagggaggttgtgaaaaagacggatttatctgaacgtcaggttgaacgatggctcagaagacgggtcaagttagacacgccaagcaccatgcaaaaattcaccgagtgcagctaccatttcttattctactgtgcgatgtttttctatgggctttacatactctgggataaatcttggttctgg"
## 
## $comp22144_c0_seq1
## [1] "tatctgatagctcgtgtagaaggaaaagtgttcacaagctattttacatcctccacccctctgggatccaccataacattaaagaaaattattggatcctcctgcctcaacaatatatgcacatctacaaatggttatgaaacactgcacaaagtttcaagtttatcctataagtcattaaggaggagaagacatacttttctaaagtatggccttgttagaaggtatacacatatgaaatatgaaagccctagcattgaacattcaaaataaatggccaaggttaaagtttttgtagacaaaatctccaatgcaatttattaagaaaggaatcggtttaaatgccatttgttttcagtgacaatgtacagtggttcatctgataatgatcaaattaactgactgcttccaatattcatacctctcagatctaaagaaaacagtattgtttaatacatttcttaataacatctaattctgccatcaaaagcttgatctttgatcaagtgttggtaacaaataaatagaataagttatcaagttacatggatatgtggacacacatgcactccttgtcaactcccctttttctgaagatgttcagtctgagtcggtgtaagaacgagacttacaatcactatacaattttttatttccaattcagttttgcatgcactctgatgttgatccatttcattttttctttctttcttctgtgcatcgtggacagaaccatttacctttgggcttcaccgtcaacccaacacaagcgaagtggaaccactcaatggggcaatccgggttatcacagccaatcatttcgccataagacacttggtgacaaagacagtaggttggttcattgggatcaacaggcatgtccagaacatctgaggggtgtgtgatggacaatgtcgacaagattggtattgatgataccacctcttctggaggactttgctggcctttcttctttttctttttgggaatttcatcatcatattcttcctttttccttttctttttgtctttatctttgttctttttgcctgaatctgagtctgctttgagcttagcagctgagctcttgtctttcagatctgcttcaaaccttgccaagtcagcatctagctttcgtatatgtttgtcaaccatttcatatgtctgcatggctagctgtactttgtcatcaccatattccctgcttttggagaatttgccttctatagttgttagtttttgggaccgtttctctgatgacattcctctgacattttttaagtattcttcagataagtggtcaatgtctttcaaaagatcttgagtgcgctggtctaaatctctcataaggttgaagtttctttgcaactctgtgggcaatgtttcaagactgtccaagtagtgttccaagtacattgctgtcgccatatttgctgtttaagagg"
## 
## $comp22403_c0_seq1
## [1] "gatcgccgtatgcgcatgtttgtttatttcgacaagcgacagcgacgtccacggtccagaactttgtctagaccaggcctatatacatttacacagagtgattatatgtgactggtattaagcatactacaatggcatgcgcacaagaggaccgccttatgacggccgccatagattttggcacgacatactcaggatactgcttctcgtttcgacatgatttcgagactgaccctctcaaagtgtcagccaacacgtggaccgcaggcactgcaggtcttgtgtccctaaaaacgcccaccactgttctcctggacggagatggaaaattagtgacgtttggttatgaagcagaagatcgctactcggagcttgcagacgatgacgaacatggcgactattattatttcagacggtttaagatgacacttttcaattcttcatcaagactaactagggataccatgttaaaagacataaccggaggaaaacagatgaaagccatcacggtttttgctcatgccatccggtacttaaaagaccacatgctgaaaaccctggatcagcgtggagccggaatcaaagccagggacatcaactgggtgctgacggttcccgccatctgggacgatcccgcgaaacagttcatgcgtgaagccgcagaggaggcaggtataaatcgagcgcagctgatgatttccctggaacccgaggcagcctcgctgttctgtaaatatctacctatagagaagttacagggcgtagataaaggaatcggcgcattcaaaccgggtagccgatacctcgtcttggatgctggaggtggcacggtcgacatcacagtccatgaggttcagttcaacggaactctgaaggaattggataaggccagtggtggcgcatggggaggtactcgagtcgatgaatccttcaaggaaatgctggaagaaattgtgggagcgggaatgctcgaggagtttgccttgtcacacacagcagactacatagatttgttccgagatttcgagacaaaaaagcgcaatgtgaaagacgacgccccaggaaagatcacaattcgtattccgataagtctgcaagagttgtacgaggaaaggtcgggagaggaaattaaaaagaaaattcgccagacgaaatacaaagatgatctcacttgggtcggcgatcgtctgcgcattaacaagccatgttttgtaaacttgttttctgcagcctgtgatggtatggtggatcacgtaaagaaattgttacgttcgtccaaagtccgaggtacaaataacatcctcatggttgggggtttttcagaatcccccttactacagaaaagaattcgtgatgaatttagtgattgtcgaataattattccacaagaagcagggttagccgtccttaaaggtgccgtcatatttggtcatcaaccagcaaccatcgcggcacgtgtagctagatgcacgtatggtatagaaaccaacacgtcctttgatgctagaaaacatcctcagtcaaagaaaaaagtggtagaaggcaaggagaaatgtaaagaagtgtttgacaggcacgtgtccattggacaagtgttggaagtagacgaagttcaaagtgaaaaaagctactggcctctctattcaaaccaaaccaaagtatcgctaccggtatattcatcctcaatggacaacccggacttcgtggacgaggctggatgtagttatctaggaaagctcacagtggatatcccgcaatacggttccgacaaagaagtgtcgacaaatttcatctttggtggtactgaactaaaagtgcaagctattgtgaaaagcacaggggaagttaccactgccaattttgactttttggaaggagacagttaagacctttcgatatgaattctactaatttttaagacatgtatatcaatatttaaaaatgatgcagcagtcataatgatagtcacagcaaatatgtaaacaacaccgctgattatcaaattacacttatttgtaaataggaaatgtaggatagtgtgtgtaatcttattaatataatacgagtaaaaaca"
## 
## $comp23253_c0_seq1
## [1] "tggccgtgaaaacggccgtttgttattataggttcctgcaagcagactttttacttgctggggtttttctgggtcttcttggggaggagcacagcctggatgttgggcaggacaccaccctgggcgatggtcacgccggacagaagtttgttcaactcctcgtcgttgcggatggccagctgcaaatgacggggaatgattctggtcttcttgttgtcacgggcggcgttgcctgccaattccaagacttcggcggccaagtactccagaacggcggccaggtacacgggggcgccggctcccactctctcggcgtagttccccttcctcagcagacggtggatacgacccacgggaaactgaagtcccgcacgggatgatcggctctttgcctttcccttcacttttcctcctttaccacgtcctgacatcttgtttttactgttgtgcgctgatgaattcaaa"
## 
## $comp23265_c1_seq1
## [1] "cctttgtttggtttaaccagattttaaaaagccctgatttcatttttgaaatcagatgggtgttgtgaacatagatctttctagtctgatctatgacgaatgacctcaacagtttagtcggtgtcatattcaacaaactgaaagactcccagccttgtggatctttgatccagccacactacaaacgaaaacaaacgcgaccattttctccctctatccttttctctcgccatcttttgcctttaagagcaaccacaccgcacgactcgcattctcggtaatgttgttaactgtaatctagaagtatggtcaaagtacagcattcttaagtttgccattctaacgggtgaacaacaaggaccaggattatttccgatcattcttgactcgggaacctgttgtacaactgacgaatgtccgtggttactaagagcctcgtttcgacattctcccgcgcagtagtaggcgtcataccatttgggtgcaatgacgaaatcccaaccgaattctatgaagctaacggttaaaggatatcgacagcaagcctcaaactgattctgctcgtcgcacgtcaaagctgtcgatcgttttcgacgaacgtgggctgtggttgtcgtacgtaaatccaagttaggttcatatccttcgtttattccagtggtgggtggaagaacaattaaattttcattattagcatcgaacgcctccaccaccagccccaagttagtagaaggatccgaagcccacttatgggccaaaccagtaatatcaaaa"
## 
## $comp23747_c0_seq1
## [1] "tcaaaacagattttggagtgtaacgtctttaaaaattactaatacagcaacaatgaaatcattttgccatgaattgcttcacgatttgccagaacagcttctggccatttctattcattgtccactctacatgagatgctgacttgagaagcaccttcagagattgattaatatgttcagaacgaatatcttgtaatatgatcagcaatagcttgtcaccgcctccgtcagccaaagtgtggttggcaatggcggcttcgtacttacaccattgatcgtccaagaaattattggacagcaccaaaatgaattttctgctgacttcaatgctttccagaaagtcatccacaaagattccacccggcaagatatctctctcgtgcaaacaaagcttgtacttgtttctctctaccatctgaacaagttcagacattatccacttcctgtcgttggtgttgtaagcaacaaaaccatcatagaggaattcatcatctgtaaatcttttataccccatgggttttctgtttttgcatgtgtatatataatacttaatgtcccaacgaaactttctcaacagaacaatggtcaaaatcaccaaggacactaatgcaaacgaagcgcatactagaatgacataggggcttattttgtgacattcggacgcaggatcaaagtgtgttatactttttcctttcaagtctgacggggaagcacagatgtattgatgaggatatccaacagtcctattttggttgttttcaacccagagtcggaaccattccagttggcatccacagtccaatggattcaaggacacatcaattcgaaaatcttttttcttccaaaggaaagcaggcagagaagattcattgatactgctcaaacgattgccacgaaaaatcaattcattcaatctagtcaaattttgaacaccgctctttcgaatttctgttattcggttaacactgtagtcaacggacgttagctgtgaaaaatcagtcagtgtagtcatttgatcgatttcgccattaacaatagccaaaactgacagatcctttagaggtgatagcatctctgcaacatcagtgttggatagatcggtgttcattattttcaatgcttttatatttctacattcagaaaagatgtatttgaagcctcctacgcttttttccaccatacgtccgatagagagtgttcggagggaattggatgataatgacctaccatctaaagcgactccatgcaagttatcaatgatcaaagtttgcaaacgattcatattcaaaaaggaatacaaggaaatgtcatgaattgatgtgtgtgaaacattaaaaaattcaagggatttcaggcagtagccctcttgtatgaaatttgttgtttcccgaataccattcactgccaggtgcaattgttgcaagtttgggaaggcggctatacgtttctcattggataaacaaaactcgggaatcttctcaaaagaattgttagtaaaatacagttcttttaaagaaggaactgagctatttggcaatttataccttgagattaggttgcttcctaaatcgattttgtggatattgattaatttagcgaaggatttaaaag"
## 
## $comp24065_c0_seq1
## [1] "gtgaacgtatataaacgaacttcatttactcggctttaacagtggtatcagagcaaagtctgaagtttgtattgagtttcagcatgaaagcgattgccgcttctttccttcctgtatttctattgttcttggccaattcaaatgccgaggaatgcgcatgtgccactaccaacgttcacgtcagatccggtgctggtgtaggtcacggaattctgggaactttaagcacagattcttgcctcgccttcaaaggtcacctgacttctgtcaatggagctcattgggccaacgtagactataacggacaggatggttggatctcatctacctacctcgcattcaaaccatgctcgttatcaggatctcataacagcggcgcaggcacgcattctacttccggctgtcccccgatcgtaagtcgaagtgaatggcatgcgcgtgcgcctaagcatcacattggtcctcttccggcagtgcccggaaacgtatacattcaccatggcgccacgcacggctgtttcacaaaggcagactgcatcaagctggtgcagtcctatcagaattaccatatggacacccacggttggccagatatcggatacagcttcatcattggagaagacggaaatatctatgaggggcggggctggaacactataggggcgcatacatataaccacaattatgatggtctaggattctgcgtcatgggagacttcacctcgcaccaaccaaacgccctagctataaacgcagtgaagagcctgattagctgcggagtaaacagcggaaggataactcataactacgtgttgaaaggtcaccgtgatgtcgggcaaaccagttgtccaggggacaaattctatgcccttatcaagacatggccgcattatcatcattgagggattttcactgaagaccctatgattgtacaatgtaatttataaagcaatcaatgacacatttgctcttcattacttttcatacacag"
## 
## $comp24195_c0_seq1
## [1] "gtaaattctatttcaccgtttccataaaatcggccaatggatcctaaaggaatagatacccacatcaataagctctcccatcttaatttggaaactgatggcggtgcatgtgttagcgaaagtaatagatatcaagaagaagcatatgattcagcgtatttttctgcttctttatctgagtttccttccactgagtccacaattaaaacagaaatcttaccacaagatcattatcccgtaccatccatcatttgctctccacatgtaagtgctcaggccacagaaactgcacaagccacagaaactgcacaaggttctcctcggtcttctcttcaactgcagacttcacgatgtcactttataccctatgttacgacacaagaaagaaactttcagaatatcaaaatttcggatccaaatgttcaaaaaatgatacatgctgacaaagacggcgatacacagctccatatggcgatcatcgaaaaccatgtttctgttgcaatttacataatacggtgtctggccgctgtagatgtcaatcttctgaaccatcaaaataacttacttgaaacacctctctggttggccatttacgaacaacaattagatgttgttgagatgttgattagaagtggcgcagatgttactgctgcagatagaaatggcgatactccgttgcatgttgcctgtaaatatggacgagatgaagcccttgatattcttcttcaaaatacaaaagcaacaactgtcaggcattgcctatctcttccaaattacggtggacaaacatgtctccatttagctgcagacagaatgcatttttctgtcataagacttctgtttagatatggaatggacgtaaatgaaaaagagaaagttcacggaatgacaattttacacaaggctgcaaaattagggaataaggtattgttagaattcattttgaatttacagggattagatttgaacacagaaacttatgctgggtatacccctattttgttagcttactttgaaggtcattttgatatcgttcttttactcaaacaagagggttgcgattttcaagatctgtcttagcaatgtgcaatgttttgcttttcactggaaatattacgtatttctatttgaatggctcggtaggagggaagttctttgggtaacaccgaaagacagagtgaaaagaagtatgtctattttgatatgctgccacttgaattgtaat"
## 
## $comp25000_c0_seq1
## [1] "ttttttttcagttttgtaatctacatttattcttcaacttcactttgagattaccactacaataggcaaccttactaagtctcccaaagttataaaagagagatggtttttaaaatttatttatgataaatggacatgaaaataataaaactcgacacaaacattgacgctgtactattaaaaccaaaagcgatggagttgatgaggtgaaagatgaggataaaaaatgtcttctaagttatatgacagtcatagtctctatatataaacttttacacaacattcaggtagagtcttttgttcagctcccagaattttctcaccatgaacactttaagctgctttctgtgtgcccttctttccaatgagagatttgtgaatatgtggaatgacaccaccaccggcaattgttgctttgattaaagaatccaactcttcatctcctctgatggcaagctgcaagtgacggggtgtgatacgctttaccttcagatctttactggcgtttcctgccaactccaacacttcagctgtcaagtactccagaatagcggcactgtacacggcagctgtggctccaacacgtccatgacttgtggtcctgttcttcaaatgtcgatgaatacgtcctacgggaaactgaagacctgctctggccgaacgagaaactgccttggccttcgcttttccggaatctttacctgctttacctcctgccatctttgctgcctgatgaaatcaatctcaaaccgacgctaacgaaggccgccaaagtatatgccagttcaaggtcacgtgataaaataaaccaatgggtacaccgcaaatgaaaacaaagga"
## 
## $comp25313_c0_seq1
## [1] "atggtaaccctgaaaggcaggttacccacgagttatttgcccctgatccaaaataaaattaagtcgtacgtgtgtcatttactcaaaccgtagaaactgcagtgtgtgcacagggactcgtcacgaattgacctcattaaaatatagaatagacggtgtcaaattttaataagacaaaaagttcgtgacgagcccctgtggccagagctcccatcccctttactagctccttcaacataactgtcagtataagaccctggtacttattatacttggaacttttgctaaactggtcagtatcttcattatatacatgcaatgagaatggatctaaatcatgcattgacatatagagtatccatactaaactacccctatactggcctatagtgaccagcccacctagtgtgacttctggtgaaatattgagcccgaatataaacgataaattttcttcaacacaattactgacgagcagatgtaccgaatacaactatcccataatagctagagttacgataactaatttgattttaaactgagaaggtatttctaaatgaggaaatttgacaaacatctatgattctgtacatgtttctgtttctttcatggaattggtatgttacttgtgtcaactttaactttgatgaatattgtatcttctttgagatatgtctttgtttcgagcacactatgggagataaaaagaggacatccagtggcaacattcatctctgtagttggtctcttgaagctgctacttgttgtgtcaggctggaaagtatcacagacatgtcttctgttgaagtcttgatccaacagcatcagggaaactttctgctggaaaggccatggaagcagggcatcaaactctcctctcatcaccacaaagaacaaggacagatgagtgcctttacccattccatccccattcaggtacactctggcacacatcttgtagccagtgcgacttgtgtaaaatggctggctgtacagggagagggttctcccacttttggcctcctcctttcttctggagtaatctcttatcttccacattaaaatgccatcataactagcagtctccagcacttggaatctcaggtctaactcagcaagcctaatgtcctggatcgccatctgcttctcattactcttcagttgattcctcacctcttgagacacccctgcctgtgaccctccacctccgggccccgctcgctctagctgagttatctgctcctgactgactcgcaagtcctctctcagcacatttaaaacttgctcctgcttttcaaggacttctcgctttggctggtgttcaatcaacctctccaaatttattaacctttctgtcaatgacactaacttttttttcatatcctttgtgatcgatttcactaagggtgtaatttcttcttttaacctttgattttcctgatgcaattcttgaacaattctttgcatttctactgcctctgtcttgcttttctcaatctcagtctgcagagcttgattctgatcgcttgtttgattggaaaatgacataactaacatcagatggtgatctgtcgctgttgtctgatgcttctccacttctttgactacatcaacaaactgacaccctatgcttttgtatccacaagggattgatcgttttgggcaagattgcaggtgactatccatctcacttcgacgcagtattatattttcacacccatttggacagttcagttgatattccaaacactcatggtcaatgtgactctgcatctgacagaagggaacatcccttgagcagtggctgcatacagcaattcgatggctgcactctaatttctgatgactggacagcttattctgaggtattttttcatgacattcaccacattccacgggtttaaactcgcattcctcagcgtgtatcataaggtttctccatattggtgtctctttacacccatgattgtgataggaacagaaaactggaagtaacatcatctctcgacgagtgcacatatctttatatatctcatttttggagaaagactccttacactcctcatccaactgtaggggacaggttgctgtgtcattttctccaaacaatggatcaatacaggactcacagatctgatgtccacactgtagttgcatggcattccttagtactgttccacacacagagcaaatgtatttatccgctactttattgacaaactgtggcaagtctgctgataataagcttgttcccaaagaagccatagaggaatccatgttgagatagcaaatttcagcttacgcggtattcaaaatattttaacaaaatacagagttttatatgcaaatgttttttaaatttacaagaatatttttttaagtcactgacagaagttgctatgtattcaaggttattattatatttcctattttttatcctatcacatctaatattctgtatagcaacagtaaaggaagaaatgatcaatatatcactccaaaaagaatcatgttttatccaatactgtagaaatacagttttttcatatagtgctgataatttacaacccatacacccctggctttgcaatcattttcatttttactgtatgtttctgcattcttaaacacaactcatctgaaataggcctacagtccgtgagccaagggcgtcgcttctaaaatggcgttgtactttactcattcggcgaa"

Wednesday, May 13, 2015

5 12 2015 TTh 310 Lab qPCR Results

Yesterday the Tuesday Thursday 310 lab ran their qPCRs for Actin, HSP70, and glutamine synthetase. I'm posting the results from their run here before posting it to Canvas.

Group 3-4
Amplification:
Melt Curve:


Group 5-6
Amplification:
Melt Curve:
Group 7-8
Amplification:
Melt Curve:
Group 9-10
Amplification:
Melt Curve:
Group 11 - 12
Amplification:
Melt Curve:

You can also see the data files for the groups here: 3-4, 5-6, 7-8, 9-10, 11-12. The raw data file can be found here.

Tuesday, May 12, 2015

5 11 2015 310 MW Lab qPCR Results

The 310 Students ran their first qPCR yesterday with primers they chose or Actin primers for normalization. I'm posting their results here first just to keep a record of what they found.

Group 3-4
Amplification:

Melt Curve:


Group 5-6:
Amplification
Melt Curve:

Group 7-8:
Amplification:
Melt Curve:

Group 9-10:
Amplification:

Melt Curve:

Group 11-12:
Amplification:


Melt Curve:



You can see the data for here for 3-4, 5-6, 7-8, 9-10, 11-12. Also you can play with the raw data file here.


Monday, May 11, 2015

5 8 2015 cDNA creation and Primer Check

On Friday I created cDNA using a complicated system calculate concentration and volume of RNA needed to produce equivalent concentrations of cDNA across the board.

First I need to get updated concentrations on all the RNA samples to calculate the volume needed for cDNA creation.

Sample IDDateng/ulA260A280260/280260/230
42715ST15/8/2015385.829.6465.0761.90.94
42715ST25/8/2015287.17.1783.9291.830.71
42715ST35/8/2015267.756.6943.6231.850.89
42715ST45/8/2015322.488.0624.3241.860.77
42715ST55/8/2015244.876.1223.4241.790.66
42715HT15/8/2015172.114.3032.5021.720.57
42715HT25/8/2015323.018.0754.3591.850.75
42715HT35/8/2015141.123.5281.8081.951.5
42715HT45/8/2015177.024.4252.5971.70.59
42715HT55/8/2015294.267.3574.0191.830.72
42815SM15/8/2015110.132.7531.4821.860.96
42815SM25/8/201543.081.0770.6241.730.57
42815SM35/8/201538.870.9720.6381.520.42
42815SM45/8/2015112.832.8211.7381.620.42
42815SM55/8/201567.161.6791.0471.60.36
42815HM15/8/201552.71.3180.7581.740.75
42815HM25/8/201572.281.8071.2021.50.35
42815HM35/8/201559.051.4761.0431.420.26
42815HM45/8/201582.52.0621.271.620.34
42815HM55/8/2015128.423.211.9111.680.4

Per Sam's instructions on the genefish wiki:
  1. Use as much RNA as possible (up to 1ug); max volume of RNA = 17.75uL. Generally, identify the RNA sample with the lowest concentration and multiply by 17.75uL. Use this quantity (ug) of RNA for each and every sample.
  2. Transfer calculated volume(s) of RNA to 0.5mL snap cap tubes or PCR plate. Adjust volumes of individual samples to 17.75uL with H2O.
  3. Add appropriate amount of primer to sample. Use 0.25ug primer per 1ug of RNA in sample (= 0.5uL of Promega oligo dT Cat#C1101 in this example). Total volume (RNA + primers) should equal 18.25uL.

Sample SM3 was the lowest concentration on the table. To use a max of 17.75 ul of RNA I could only produce 690 ng of RNA with SM3. I set the bar to 690 ng and made the following calculations of RNA, Water, and Primer needed. SM2 and SM3 still ended up producing a volume larger than desired due to me having to use a 1:10 dilution of primer to account for the less than optimum concentration of RNA.  Instead of using the standard 0.5 ul of oligo Primer, I used 0.345 which I diluted in a 1:10 ratio. For the 1:10 dilution I added 7.6 ul of oligo primer to 76 ul of water to generation 22 reactions worth of primer. 

Sample IDng/ulul RNA for 690 ngul h20 for 17.901:10 primer ultotal volume
42715ST1385.821.7913.013.4518.25
42715ST2287.12.4012.393.4518.25
42715ST3267.752.5812.223.4518.25
42715ST4322.482.1412.663.4518.25
42715ST5244.872.8211.983.4518.25
42715HT1172.114.0110.793.4518.25
42715HT2323.012.1412.663.4518.25
42715HT3141.124.899.913.4518.25
42715HT4177.023.9010.903.4518.25
42715HT5294.262.3412.453.4518.25
42815SM1110.136.278.533.4518.25
42815SM243.0816.020.003.4519.47
42815SM338.8717.750.003.4521.20
42815SM4112.836.128.683.4518.25
42815SM567.1610.274.523.4518.25
42815HM152.713.091.703.4518.25
42815HM272.289.555.253.4518.25
42815HM359.0511.693.113.4518.25
42815HM482.58.366.433.4518.25
42815HM5128.425.379.423.4518.25
1. Added the water first
2. Added the RNA
3. Added the primer to each reaction tube. 
4. Incubated in the Thermocycler for 5 minutes at 70 C
5. Immediately transferred to ice after completion. 

Then I made up the reverse transcriptase master mix:

ReagentsVolume ulReactions X22
M-MLV RT Buffer5110
10 mM dNTPs1.2527.5
M-MLV RT0.3457.59
H200.1553.41
Total Volume6.75148.5
1.Added 6.75 ul of the master mix to each tube. 
2. Ran the following program on the thermocycler.

Amplify42 C1 hr
Inactivate95 C3 min
During the program run I picked up the new primers from the science depot in the med school. 
I rehydrated the primer stock with Nanopure H20 since no TE was available. I determined the rehydration volume based on the nanoMolar concentrations. I then made working stock from these rehydrated stocks using a 1:10 dilution of 10 ul primer to 90 ul Nanopure H20. 

Once the cDNA amplification was complete I ran a Primer/cDNA check using a qPCR and all the primers we are using for 310 lab. 

Primers:
Superoxide dismutase (SD)
Glutamine synthetase (GS)
Citrate synthase (CS)
HSP70 (HSP)
Actin (ACT)

I made a Master Mix for 6 reactions for each of the 5 primers. 

VolumeReactions X6
Ssofast Evagreen MM 1060
FWD Primer0.53
REV Primer0.53
Nuclease Free H2O848
I ran a template control and a positive control (Fidalgo seed oysters extracted on 3/23/2015 with a concentration of 167.3 ng/ul) for each primer as well as 4 cDNA samples I produced. 

Plate layout:
89101112
SDGSCSHSPACT
C-C-C-C-C-
C+C+C+C+C+
42715ST142715HT142715SM142715HM142715ST5
42715ST242715HT242715SM242715HM242715HT5
42715ST342715HT342715SM342715HM342715SM5
42715ST442715HT442715SM442715HM442715HM5
I ran the following program:

Sybr New Plate+Sybr cDNA 55 melt 2
StepTemperatureTime
Initiation95 C10 min
Elongation95 C15 sec
55 C15 sec
Read
72 C15 sec
Read
Repeat Elongation 40 times
Termination95 C1 min
55 C1 sec
Melt Curve Manual ramp 0.2C per sec Read 0.5 C65 - 95 C30 sec
21 C10 min
End
You can see the amplification curves below:

Melt Curves:

The 3 melt curve peaks correlate with HSP70, Actin, and Glutamine synthetase primers working with the cDNA. The Superoxide dismutase and the citrate synthase primers did not work for the cDNA. 

You can see the raw qPCR data here.

Moving forward, the 310 students will be running their samples using the primers today and tomorrow. I'll update the notebook with their data and some graphs to hopefully produce some interesting info about stress resilience mechanisms in the olys.