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Lisa K. Johnson
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| THIS REPOSITORY IS A WORK_IN_PROGRESS. | ||
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| Hybrid genome assembly of the killifish, *Fundulus olivaceus* with ONT and Illumina NovaSeq | ||
| Genome assemblies of four *Fundulus* killifish with ONT and Illumina | ||
| === | ||
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| # Thank you! | ||
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| * C. Titus Brown and Moore Foundation for encouragement and funding to go to Porecamp! | ||
| * [Andrew Whitehead](https://whiteheadresearch.wordpress.com/) for motivating questions, and Tony Gil for assistance with samples and hmw DNA extraction: [Agilent protocol (Catalog #200600), Protocol II: Extraction from Whole Tissue](http://www.agilent.com/cs/library/usermanuals/public/200600.pdf) | ||
| * [TAMU seq facility](http://www.txgen.tamu.edu/), Charlie Johnson, Richard Metz, Joshua Hill. Hosted Porecamp, then they offered us free NovaSeq! | ||
| * [Porecamp](http://www.txgen.tamu.edu/porecamp_usa/): Nick Loman, Joshua Quick, Mick Watson, Matt Loose, John Tyson | ||
| * [David Duvernell, Southern Illinois State University](http://www.siue.edu/~dduvern/) for generously providing *F. olivaceus* fish | ||
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| # Purpose | ||
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| To generate a genome assembly for each of several freshwater *Fundulus* killifish. In particular, *Fundulus olivaceus* data were collected for this analysis. Subsequent data will be collected for other freshwater *Fundulus* species. The final assemblies for each species will be used to compare genomic regions to brackish/marine congeners. | ||
| 2019 | ||
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| Want to be able to: | ||
| Lisa K. Johnson [1,2], Ruta Sahasrabudhe [3], Tony Gill [1], Jennifer Roach [1], Lutz Froenicke [3], C. Titus Brown [2], Andrew Whitehead* [1] | ||
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| * Scan for regulatory elements, such as osmolality/salinity-responsive enhance (OSREs) ([Wang and Kültz 2017](http://www.pnas.org/content/114/13/E2729)). | ||
| * Detect gene duplications, deletions of regions | ||
| * Potentially help to resolve RNAseq assemblies, detect isoform switching, e.g. NKCC1 alpha1a and alpha1b subunit (assemblies with only short reads cannot resolve whether transcripts span multiple genomic regions): | ||
| https://link.springer.com/article/10.1007%2Fs00360-012-0719-y | ||
| http://ajpcell.physiology.org/content/ajpcell/287/2/C300.full.pdf | ||
| http://jeb.biologists.org/content/212/24/3994 | ||
| [1] Department of Environmental Toxicology, University of California, Davis | ||
| [2] Department of Population Health & Reproduction, School of Veterinary Medicine, University of California, Davis | ||
| [3] DNA Technologies Core, Genome Center, University of California, Davis | ||
| *corresponding author: [email protected] | ||
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| Long reads can potentially help to solve problems with short-read assemblies. | ||
| # Workflow | ||
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| # Questions | ||
| 1. Do we have enough data from *F. olivaceus* to meet our goals to detect regulatory elements and isoforms? Do we need more ONT reads? More Illumina reads? Could we get away with fewer Illumina reads? | ||
| 2. What is the most cost-efficient way to generate a reference genome when there is a closely-related sister species? ONT and Illumina? ONT only? | ||
| 1. QC [scripts](https://github.com/johnsolk/ONT_Illumina_genome_assembly/tree/master/scripts/1_qc) | ||
| 2. Assembly [scripts](https://github.com/johnsolk/ONT_Illumina_genome_assembly/tree/master/scripts/2_assembly) | ||
| 3. Evaluation [scripts](https://github.com/johnsolk/ONT_Illumina_genome_assembly/tree/master/scripts/3_evaluation) | ||
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| # Data Generation | ||
| # Data | ||
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| * NovaSeq: 600 million reads of 2x150 (300b) = 180Gb | ||
| * 3 ONT flowcells, albacore base calling | ||
| Raw ONT and Illumina reads: https://www.ebi.ac.uk/ena/data/view/PRJEB29136 | ||
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| ``` | ||
| stats for ../fastq2/porecamp_killifish2.fastq | ||
| sum = 4962626713, n = 740248, ave = 6704.01, largest = 973552 | ||
| N50 = 12726, n = 117202 | ||
| N60 = 10357, n = 160433 | ||
| N70 = 8098, n = 214460 | ||
| N80 = 5724, n = 286845 | ||
| N90 = 3229, n = 400661 | ||
| N100 = 5, n = 740248 | ||
| N_count = 0 | ||
| Gaps = 0 | ||
| ``` | ||
| Johnson, L. (2019, June 21). Genome assemblies of killifish from the Fundulus genus with ONT. https://doi.org/10.17605/osf.io/zjv86 | ||
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| *Some* files are backed up on [OSF](https://osf.io/gek4p/). See [Instructions for installing osf client](http://osfclient.readthedocs.io/en/stable/). | ||
| [](https://doi.org/10.5281/zenodo.3251034) | ||
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| ``` | ||
| export OSF_PASSWORD=password | ||
| export [email protected] | ||
| # list files in OSF | ||
| osf -p zjv86 ls | ||
| # copy files from OSF to local (or hpc - wherever you're working) | ||
| osf -p zjv86 clone Folivaceus_hybrid_genome_assembly | ||
| osf -p zjv86 upload /mnt/scratch/ljcohen/onp_Porecamp_killifish/fastq2/porecamp_killifish_ONT_reads.fastq porecamp_killifish_ONT_reads.fastq | ||
| Johnson, Lisa K., Brown, C. Titus, & Whitehead, Andrew. (2019). Draft genome assemblies of killifish from the Fundulus genus with ONT and Illumina sequencing platforms [Data set]. Zenodo. http://doi.org/10.5281/zenodo.3251034 | ||
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| ``` | ||
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| 12/18/2017: Need to backup ONT and NovaSeq raw files to a location for safekeeping. NCBI, S3? | ||
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| Subset data for analysis runthrough. | ||
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| ``` | ||
| cd | ||
| head -2000000 F_olivaceus_ONT.fasta > subset_F_olivaceus_ONT_1m.fasta | ||
| zcat F_olivaceus_R1.fastq.gz | head -2000000 > subset_F_olivaceus_R1.fastq | ||
| zcat F_olivaceus_R2.fastq.gz | head -2000000 > subset_F_olivaceus_F2.fastq | ||
| ``` | ||
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| [Methods: Workflow, commands used and notes](https://github.com/ljcohen/hybrid_genome_assembly/blob/master/workflow.md) | ||
| # Thank you! | ||
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| Johnson, L. (2018, July 20). Hybrid genome assembly of the freshwater killifish, Fundulus olivaceus with ONT and Illumina NovaSeq. https://doi.org/10.17605/osf.io/zjv86 | ||
| * [TAMU seq facility](http://www.txgen.tamu.edu/), Charlie Johnson, Richard Metz, Joshua Hill. Hosted Porecamp, then they offered us free NovaSeq! | ||
| * [Porecamp](http://www.txgen.tamu.edu/porecamp_usa/): Nick Loman, Joshua Quick, Mick Watson, Matt Loose, John Tyson | ||
| * [David Duvernell, Southern Illinois State University](http://www.siue.edu/~dduvern/) and [Jacob Shaefer](http://ichthyology.usm.edu/) for generously providing *Fundulus* fish |