Reports of research work funded by grants prior to 2016
Victoria University of Wellington
P Atkinson, B Busby
Chemical Genetics Library, School of Biological Sciences
The specific objective of this proposal was to characterise the genomic sequences of three newly created gene knockout libraries (ssDMAs) derived from natural strains of yeast in order to verify the back-crossing methodology used to create the ssDMAs. Genome sequencing of pooled libraries of the three new ssDMAs was achieved by employing a commercial company previously utilised by this laboratory (Christina Roberts report to WMRF 2015), see below. Analysis of the sequencing results for variant calling is being performed by Christina Roberts derived from methods in her PhD thesis (examined and completed, 2016).
Background and method
Statins are the most prescribed of all human therapeutic drugs and are highly successful in treatment of life-threatening atherosclerosis. However, they have side effects ranging from mild muscle pains to severe muscle myopathy, liver damage, gastro intestinal upset and even neurological effects including memory loss and confusion. The severity of the various symptoms often depends on the individual receiving treatment.
Recently gene functions have been studied as extensive genetic interaction networks comprising synergistic functional interactions between pairs of genes in a widely-used technology developed by Charles Boone and colleagues (UoToronto) called synthetic genetic array analysis (SGA). Elucidation of such networks related to drug phenotypes (statins in this grant) provides additional information to classical genetics as to what genes and metabolic pathways might be involved in polygenic phenotypes. Importantly, genetic interaction networks are important to pinpointing where side-effects might arise in drug effects. We were aimed to investigate the genetic basis of individual phenotypic responses.
To generate genetic interaction networks relevant to individual responses, it was necessary to create new genome-wide deletion mutant arrays (DMAs) for each of the strains, such DMAs being the basic tool in genetic interaction network elucidation. The new DMAs, called ssDMAs, were created by extensive backcrossing of the commercially available standard yeast DMA with the three statin resistant yeast strains to create the three new genome-wide deletion ssDMAs. The aim of the backcrossing was to minimise the genome contribution from the commercially available deletion set leaving only the gene knockout cassettes comprising recombination flanking regions, the gene deletion replacement marker and identification bar-codes. The sequencing and analysis of ssDMAs was necessary to confirm whether the 6 back-crossing steps utilised by Bede Busby in his PhD thesis (examined and completed 2016) achieved the back-crossing aim i.e. was efficacious. To answer this question nine yeast strains were sequenced. These were:-
- SK1 DMA (pooled)
- YPS606 DMA (pooled)
- Y55 DMA (pooled)
- BY4242 DMA control (pooled)
- Commercial DMA (pooled)
- BY4742 Backcross DMA control parental strain
- SK1 parental strain
- YPS606 parental strain
- Y55 parental strain
Results and progress to date:
The whole-genome sequencing provided 35GB of high-quality Illumina raw sequencing data in seven samples (two were obtained from public databases). Bioinformatic analysis, carried out by Christina Roberts, was performed by alignment of the reads to the reference yeast genome using the software package Burrows-Wheeler Aligner (BWA). Variants were called using SAMTOOLS. Analysis is still in progress and will be completed by the end of August.
Funding granted by WMRF allowed us to carry out Illumina sequencing for this purpose through Macrogen Inc. in South Korea. Costs for the sequencing of ten strains of yeast was $USD390 strain ($USD3900 USD/10 strains; $NZD6200) at 80x coverage/strain. This includes: sample shipping, quality control, library preparation for Illumina sequencing (using a Truseq Nano DNA kit) and 1 Gb of Illumina sequencing per sample.
We achieved and paid for sequencing of the above listed strains. This information is being used to verify the back-crossing efficacy of Bede Busby’s newly created deletion mutant arrays (ssDMAs) and, with the temporary exception of the corrupted sequence information, is showing that the back-crossing was efficacious. Bede Busby’s ssDMAs will have many uses in the research community not the least of which the SGA procedure will be made easier. This is because sporulation efficiency of parents in the ssDMAs is ~70-80%, compared with ~3-4% of the widely used Boone DMA used in current SGA technology. One manuscript is in preparation for submission by September 2016.