An updated reference assembly and annotation Tutorial aim: The aim of this tutorial is to guide users through the process of manual editing using the range of functionalities Apollo provides to update the PN40024.v4.1 annotation which will accompany the new PN40024.v4 genome assembly on Vitis vinifera cv. PN40024. Manual curation is an ongoing task so the idea would be that the grapevine community continues to use Apollo beyond this workshop. Introduction The PN40024.v4 genome assembly is built upon the previous 12X.v2 PN40024 assembly.  The strategy aimed to improve the previous assembly without discarding the existing Sanger assembly which is still a high quality sequence in terms of base pair accuracy. The new assembly includes new PacBio and Illumina reads which allow the filling of gaps, improvement of existing scaffolds and sequence correction of newly  incorporated PacBio regions. The update shows promising improvements including a significant increase in the average size of scaffolds (235,000 to 741,572 bp), a significant  decrease in the number of Ns (~16 to ~2 million bp) and an improved assignment of unknown contigs to chromosomes (~27 to ~12 million bp). The new assembly supports better alignment of RNA-Seq reads across a range of cultivars. In addition, heterozygous regions have been delimited and provided with the assembly as smaller alternative chromosomes containing only those regions. One interesting finding is that the PN40024 cultivar was obtained through self-crossing of cv. Helfensteiner and not cv. Pinot Noir as previously thought. Cultivar Helfensteiner was initially created in 1931 from the crossing cv. Pinot Précoce Noir (early ripening PN) and cv. Schiava Grossa. The PN40024.v4 annotation was created with the aim of obtaining a more reasonable number of protein-coding genes (35,197 as opposed to 41,733) with respect to VCOST.v3 since this version contained many extra protein-coding genes which are unlikely to be real. In an effort to preserve VCOST.v3 gene IDs where possible (i.e. in cases where chromosome numbers are unchanged) a reciprocal best-hits strategy between VCOST.v3 and PN40024.v4.1 annotations was carried out. This allowed for the transfer of 67.27% of VCOST.v3 gene IDs. In general, the new de novo annotation performed better than the previous annotation in terms of BUSCO stats.  Motives for a curated manual annotation In silico strategies for genome annotation have their limitations especially with regards to the actual expression of individual transcripts, which can be shown by transcriptomic data (i.e. RNA-Seq). Although the new annotation is already an improvement with respect to the previous version, some gene families such as those derived from recent gene expansions have not been well annotated. For a better informed annotation, several data sources have been integrated within Apollo to help the user choose the best gene model for a particular transcript. A summary of the tracks are outlined below: PN40024.v4.1 annotation (v4 de novo annotation) REF  ALT (annotation corresponding to alternative heterozygous regions only) Previous annotation versions (Transfer of previous gene model coordinates to the new assembly) CRIBI V1 VCOSTv3 Gene catalogue Gene catalogue (Integrape v1.1 catalogue, P450s, RGAs and more) RNAseq Mixed samples (12 RNA-Seq studies from a range of tissues) Mixed samples – insert size <= 300 – MQ >= 40 (filtered by intron length and read quality) Mixed samples with multimapping PN40024 RNAseq on REF only (PN40024 mixed tissues) –> Organ-independent samples –> SOON TO BE INCLUDED CS IsoSeq max introns 20kb (cv. Cabernet Sauvignon showing full transcripts in fruit) Separate RNAseq (11 runs from mixed samples; the remaining run was already a blend of tissues) RCDN1_S1 (Vitis sylvestris C1-2, green berries) RCDN5_S2 (Vitis sylvestris C1-2, ripening berries) SRR1502882 (cv. Carignan, leaves) SRR2015361 (cv. Riesling, Shoot) SRR4447140 (cv. Jingxiangyu, leaves) SRR5435950 (cv. Pinot Noir, leaves) SRR6195043 (cv. Vlaska, petioles) SRR6365708 (cv. Bangalore blue, leaves) SRR7192360 (cv. Cabernet Franc, leaves) SRR7451534 (cv. Victoria, leaves) SRR7768546 (cv. Rosario Bianco, buds) Orthologs (Transferred gene models from different databases) Arabidopsis ORTHODB (orthologous protein-coding genes across vertebrates, arthropods, fungi, plants, and bacteria.) SWISSPROT (protein database) Vitales SNPs PN40024 Illumina SNPs (SNPs identified by Illumina reads) Others (Useful during annotation as a gene that crosses a scaffold or an N gap is problematic) Heterozygous region Scaffolds N gaps Repeat regions