In ipb-halle/GoldenMutagenesis: A tool to calculate and validate primers for Golden Gate Cloning

Quick Quality Control

You can find the lastest version of this file at https://github.com/ipb-halle/GoldenMutagenesis/blob/master/vignettes/QQC.md

knitr::opts_chunk$set(echo = TRUE)

Experimental Workflow

Target sequence

open reading frame YfeX in pCA24N (Chloramphenicol resistance)

Clone into

seperate gene fragments into pAGM9121 first\ then reassemble into pAGM22082_cRed

Genomic sequence YfeX

ATGTCTCAGGTTCAGAGTGGCATTTTGCCAGAACATTGCCGCGCGGCGATTTGGATCGAAGCCAACGTGAAAGGGGAAGTTGACGCCCTGCGTGCGGCCAGTAAAACATTTGCCGACAAACTGGCAACTTTTGAAGCGAAATTCCCGGACGCGCATCTTGGTGCGGTGGTTGCCTTTGGTAACAACACCTGGCGCGCTCTGAGCGGCGGCGTTGGGGCAGAAGAGCTGAAAGATTTTCCGGGCTACGGTAAAGGCCTTGCGCCGACGACCCAGTTCGATGTGTTGATCCACATTCTTTCTCTGCGTCACGACGTAAACTTCTCTGTCGCCCAGGCGGCGATGGAAGCCTTTGGTGACTGCATTGAAGTGAAAGAAGAGATCCACGGCTTCCGTTGGGTTGAAGAGCGTGACCTGAGCGGCTTTGTTGACGGTACGGAAAACCCGGCGGGTGAAGAGACGCGTCGCGAAGTGGCGGTTATCAAAGACGGCGTGGATGCGGGCGGCAGCTATGTGTTTGTCCAGCGTTGGGAACACAACCTGAAGCAGCTCAACCGGATGAGCGTTCACGATCAGGAGATGGTGATCGGGCGCACCAAAGAGGCCAACGAAGAGATCGACGGCGACGAACGTCCGGAAACCTCTCACCTCACCCGCGTTGATCTGAAAGAAGATGGCAAAGGGCTGAAGATTGTTCGCCAGAGCCTGCCGTACGGCACTGCCAGTGGCACTCACGGTCTGTACTTCTGCGCCTACTGCGCGCGTCTGCATAACATTGAGCAGCAACTGCTGAGCATGTTTGGCGATACCGATGGTAAGCGTGATGCGATGTTGCGTTTCACCAAACCGGTAACCGGCGGCTATTATTTCGCACCGTCGCTGGACAAGTTGATGGCGCTGTAA

Restriction Enzyme

Level 0

BbsI

Recognition site: GAAGAC

Level 2

BsaI

Recognition site: GGTCTC

Envisioned Mutations

Aspartic Acid - 137\ Aspartic Acid - 143\ Asparagine - 147\ Arginine - 232\ Serine - 234\ \ Substitute for NDT

R Workflow

Sequence and Mutations

The target sequence and the envisioned mutations are used as input again.

library("GoldenMutagenesis")

input_sequence<-"ATGTCTCAGGTTCAGAGTGGCATTTTGCCAGAACATTGCCGCGCGGCGATTTGGATCGAAGCCAACGTGAAAGGGGAAGTTGACGCCCTGCGTGCGGCCAGTAAAACATTTGCCGACAAACTGGCAACTTTTGAAGCGAAATTCCCGGACGCGCATCTTGGTGCGGTGGTTGCCTTTGGTAACAACACCTGGCGCGCTCTGAGCGGCGGCGTTGGGGCAGAAGAGCTGAAAGATTTTCCGGGCTACGGTAAAGGCCTTGCGCCGACGACCCAGTTCGATGTGTTGATCCACATTCTTTCTCTGCGTCACGACGTAAACTTCTCTGTCGCCCAGGCGGCGATGGAAGCCTTTGGTGACTGCATTGAAGTGAAAGAAGAGATCCACGGCTTCCGTTGGGTTGAAGAGCGTGACCTGAGCGGCTTTGTTGACGGTACGGAAAACCCGGCGGGTGAAGAGACGCGTCGCGAAGTGGCGGTTATCAAAGACGGCGTGGATGCGGGCGGCAGCTATGTGTTTGTCCAGCGTTGGGAACACAACCTGAAGCAGCTCAACCGGATGAGCGTTCACGATCAGGAGATGGTGATCGGGCGCACCAAAGAGGCCAACGAAGAGATCGACGGCGACGAACGTCCGGAAACCTCTCACCTCACCCGCGTTGATCTGAAAGAAGATGGCAAAGGGCTGAAGATTGTTCGCCAGAGCCTGCCGTACGGCACTGCCAGTGGCACTCACGGTCTGTACTTCTGCGCCTACTGCGCGCGTCTGCATAACATTGAGCAGCAACTGCTGAGCATGTTTGGCGATACCGATGGTAAGCGTGATGCGATGTTGCGTTTCACCAAACCGGTAACCGGCGGCTATTATTTCGCACCGTCGCTGGACAAGTTGATGGCGCTGTAA"

mutations<-c(137,143,147,232,234)

Quality Control

The functions aligns the obtained sequencing results to the target gene sequence. It also tries to align the reverse complement of the obtained sequence. Afterwards it checks for mismatches between the sequences. Mismatches are likely to be sucessfully mutated nucleotides. Positions regarded as mismatches are displayed as pie charts. The shown distributions are based on the signal intensities of the four nucleobases at the mismatch positions. You can compare the pie charts with expected pattern of randomization, therefore validating the quality of the created library.

Forward Sequencing

abfile<-"sequences/Yfex_0activesite_for_EF01147142.ab1")
base_distribution(input_sequence=input_sequence, ab1file=abfile, replacements=mutations)

abfile<-system.file("sequences", "Yfex_0activesite_for_EF01147142.ab1", package="GoldenMutagenesis")
base_distribution(input_sequence=input_sequence, ab1file=abfile, replacements=mutations)

Reverse Sequencing

abfile<-"sequences/Yfex_activesite_rev_EF01147143.ab1")
base_distribution(input_sequence=input_sequence, ab1file=abfile, replacements=mutations)

abfile<-system.file("sequences", "Yfex_activesite_rev_EF01147143.ab1", package="GoldenMutagenesis")
base_distribution(input_sequence=input_sequence, ab1file=abfile, replacements=mutations)

ipb-halle/GoldenMutagenesis documentation built on Sept. 1, 2020, 3:24 p.m.