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Update on the assembly and annotation of the blueberry genome
- 1. www.P2EP.org THE CURRENT STATUS OF THE BLUEBERRY GENOME Robert Reid rreid2@uncc.edu Department of Bioinformatics & Genomics University of North Carolina Charlotte BLUEPRINTS FOR BLUEBERRY
- 2. www.P2EP.org 2009~ 76 BP Illumina GA II sequencing 3Kb & some 20KB 454 pyrosequencing 36 BP Illumina sequencing 2011 454-pyrosequencing 8 kb and 20 kb paired-end insert 2013 Illumina Hiseq (5 lanes) Illumina Nextera paired-end sequencing Vaccinium.org website (WSU) 2014 Masurca and GARM assembly BAC libraries (UF), BAC-end sequencing (NCSU) 2015 SSPACE (modified) assembly Gene annotation, RNA-Seq (Gupta et al., 2015) Repeat annotations, map alignments BLUEBERRY PROJECT TIMELINE
- 4. www.P2EP.org Much room for improvement still **Estimated genome size = 608 MB (Costich et al., 1993)
- 5. www.P2EP.org MARKER ALIGNMENT TO SCAFFOLDS Linkage Map # of markers # of scaffolds Size (bp) *Tetraploid - Draper 689 358 121,530,818 *Tetraploid - Jewel 576 328 112,427,224 Interspecific hybrid 322 190 74,069,152 Diploid 318 153 56,781319 Cranberry 138 40 15,934975 696 scaffolds were assigned to at least one linkage group, the total size was 214 Mb *earlier version of map markers than what was published
- 6. www.P2EP.org GENOME COMPLETENESS Missing gene duplicate complete fragments BUSCO2 CEGMA1 (1645 Core genes) 48% 22% 18% 12% (458 Core genes) 2http://busco.ezlab.org/ Match No match 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% Newbler (454 reads) Nextera hybrid Assembly Nextera Plus BAC end sequencing 356356350
- 7. www.P2EP.org Gage identifies 84 KEGG pathways from gene predictions. Top pathways found: 1. Pyruvate metabolism 2. eta-alanine metabolism 3. Ribosome biogenesis 4. RNA polymerase 5. Pyrimidine metabolism ANNOTATING PATHWAYS VIA GAGE Luo et al., GAGE: Generally Applicable Gene Set Enrichment for Pathways Analysis. BMC Bioinformatics, 2009, 10:161 Predicted genes Predicted proteins Align to Ref- Seq Map to KEGG Identify most abundant KEGG pathways RNA-Seq Gene prediction tools Augustus Genemark SNAP (100,000 genes) transdecoder BLASTP To Grape OR Potato NCBI REFSEQ GAGE/ pathview GAGE
- 8. www.P2EP.org MAPPED TO GRAPE vvi04141 Protein processing in endoplasmic reticulum vvi00510 N-Glycan biosynthesis vvi00350 Tyrosine metabolism vvi00561 Glycerolipid metabolism vvi03020 RNA polymerase vvi04120 Ubiquitin mediated proteolysis vvi03022 Basal transcription factors vvi00950 Isoquinoline alkaloid biosynthesis vvi00030 Pentose phosphate pathway vvi00730 Thiamine metabolism vvi00960 Tropane, piperidine and pyridine alkaloid biosynthesis vvi00500 Starch and sucrose metabolism vvi03420 Nucleotide excision repair vvi00196 Photosynthesis - antenna proteins vvi03060 Protein export vvi00565 Ether lipid metabolism vvi03430 Mismatch repair vvi00770 Pantothenate and CoA biosynthesis vvi00071 Fatty acid degradation vvi00380 Tryptophan metabolism vvi00520 Amino sugar and nucleotide sugar metabolism vvi00600 Sphingolipid metabolism vvi00040 Pentose and glucuronate interconversions
- 9. www.P2EP.org MAPPING VIA PATHVIEW* *Luo W, Brouwer C. Pathview: an R/Biocondutor package for pathway-based data integration and visualization. Bioinformatics, 2013 Predicted genes Predicted proteins Align to Ref-Seq Map to KEGG Overlay onto KEGG pathway RNA-Seq 1M isoforms Gene prediction tools Augustus Genemark SNAP (100,000 genes) Transdecoder BLASTP To Grape OR potato pathview pathview
- 11. www.P2EP.org 2 AVAILABLE ONLINE ANNOTATION RESOURCES https://www.vaccinium.org Anne Lorraine http://bioviz.org/igb/index.html Dorrie Maine - WSU
- 12. www.P2EP.org FUTURE STEPS FOR GENOMICS Improving the genome Massimo Iorizzo Hamid Ashrafi Jeannie Rowland Improve contiguity Resolve repeat regions Fill in the gaps Improving the linkage maps Higher density map More anchoring points Optical map To improve scaffold / contig ordering
- 13. ACKNOWLEDGEMENTS Allan Brown CGIAR Ying-Chen Lin NC State Mary Ann Lila NC State Raad Gharaibeh -UNCC Rachel Walstead -UNCC Gregario Lingchanco-UNCC Cory R Brouwer -UNCC Jeannie Rowland USDA-ARS Dorrie Maine - WSU Garron Wright DHMRI Mark Burk DHMRI James Olmstead U of Florida And many more
Editor's Notes
- #2: P2EP started approximately 3 years ago and is a collaboration between universities and industry studying the nutritional qualities of blueberries, broccoli, oats, strawberries, pineapple and bananas. The program has a major educational component as well and last summer we had 40 grad, undergrad and even a couple high school students actively participating in nutritional research.
- #3: The blueberry genomic project in NC State has been actively carried out for several years. Here I want to when and what we have done on this project. Before 2011, we've already have some Illimina short sequences, which were only 36~76 basepairs, we also have long sequences from 454-pyroseuqences. I came to the United States and joined this project in 2012. During these three years, so far, we did one more sequencing using Illumina, and created several version of assembly with different assembler software. Before this April, Newbler assembly was the primary assembly we used for this project. But after we got the BAC-end sequence from BAC libraries, we were able to re-assemble the scaffolds, putting some small pieces together which was really difficult when you only have fragmented scaffolds. Then, now we have a more SSPACE assembly that contained much larger scaffolds than previous one.
- #12: For all the species that have been studied well in anthocyanin biosynthesis, grapevine (Vitis vinifera) is suggested to be the closest related species, which just published early this year, so here we used grape protein sequences as reference to search in blueberry genome. The first step was to search grape's protein sequences in KEGG pathway (Kyoto Encyclopedia of Genes and Genomes), then blast against blueberry scaffold data, which is stored in genome database for vaccinium website. From the blast results, we can know which scaffold contained putative genes, and start/stop point of the genes, also got the blueberry protein sequences.