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Sequencing

Genomic DNA was extracted from leaf tissue of a single Mediterranean olive tree (Olea europaea L. subsp. europaea var. europaea cv. 'Farga'; NCBI Taxonomy ID: 158383). This tree, named ‘Santander’, was translocated from the Maestrazgo region (Eastern Spain) to Boadilla del Monte (Madrid, Spain) in 2005. O. europaea is a common tree in Spain and there are no legal restrictions on its use for research, including cv. Farga.

The tree age was estimated to be 1,200 years old based on dendrometric analyses (Antonio Prieto-Rodríguez personal communication). A combination of fosmid and whole genome shotgun (WGS) libraries were sequenced using Illumina sequencing equipment.

The standard Illumina protocol was followed, with minor modifications to create short-insert paired-end (PE) libraries (Illumina Inc., Cat. # PE-930–1001), which were run on different types of Illumina sequencers (MiSeq 2×250, 2×300, 2×500, 1×600 and HiSeq2500 2×150) according to standard procedures. The MiSeq XL modes (2×500 and 1×600) were carried out according to the MiSeq modifications reported in [1] and with the technical support of Illumina.

Primary data analysis was carried out using the standard Illumina pipeline (HCS 2.0.12.0, RTA 1.17.21.3). Mate-pair (MP) libraries (3, 5, 7 and 10 kb fragment sizes) were constructed at the CRG sequencing unit according to the Nextera Mate Pair Preparation protocol (Illumina Inc.), and sequenced on the HiSeq2500 platform in 2x150bp read length runs. The number of lanes and raw sequenced outputs for each library are summarized in Table 1.

Preliminary kmer analysis of PE data (Fig. 1) indicated a high level of heterozygosity in this sample. To reduce the risk of separately assembling two different haplotypes from the same locus and including them in the final assembly, a fosmid pooling strategy was chosen similar to the one used for the oyster genome project [2]. A fosmid library of 155,000 clones was constructed in the pNGS vector (Lucigen Corp.). Ninety-six pools of ~1,600 clones each were made, and the purified DNA was used to construct short-insert PE libraries using the TruSeq™ DNA Sample Preparation Kit v2 (Illumina Inc.) and the KAPA Library Preparation kit (Kapa Biosystems) according to manufacturers’ instructions. The pools were sequenced using TruSeq SBS Kit v3-HS (Illumina Inc.), in PE mode, 2×150 bp, in a fraction of a sequencing lane of the HiSeq2000 flowcell v3 (Illumina Inc.) according to standard Illumina operation procedures. The raw sequence yield per pool was 11.3 Gb on average (SD: 2 Gb), corresponding to ~150 × depth. In addition a fosmid-end library was created from the same set of clones using the Lucigen pNGS protocol and run in one lane of a HiSeq2000.

RNA was prepared from seven different tissues or developmental stages (root, young leaf, mature leaf, flower, flower bud, immature fruit, and green olives), using the Zymo ZR Plant RNA extraction kit (Zymo Research, Irvine, CA). Then, RNA-Seq libraries were prepared using the TruSeq™ RNA Sample Prep Kit v2 (Illumina Inc.) with minor modifications, and libraries were sequenced using the TruSeq SBS Kit v3-HS in PE mode with a read length of 2×75 bp. Over 50 million PE reads per sample were generated in a fraction of a sequencing lane on a HiSeq2000 (Illumina Inc.), following the manufacturer’s protocol. Image analysis, base calling and run quality scoring were processed using the manufacturer’s software Real Time Analysis (RTA 1.13.48), followed by generation of FASTQ sequence files using CASAVA software (Illumina Inc.).

Genome assembly

A kmer analysis was performed to estimate the genome size, level of heterozygosity and repeat content of the sequenced genome. Using the software Jellyfish v1.1.10 [3], 17-mers were extracted from the WGS PE reads (PE400), and unique kmers were counted and plotted according to kmer depth (Fig. 1). The homozygous or main peak is found at a depth of ~52x. The estimated genome size (found by dividing the total number of kmers by the kmer depth of the main peak) is 1.38 Gb, which is at the low end of the range of empirical estimates. The C-value ranges from 1.45–2.33 pg (1.42 Gb–2.28 Gb), with the median at 1.59 pg (1.56 Gb) (data from [4], see [5–9]), suggesting the existence of variation in the repetitive fraction of the genome for the species. The left peak at 26x kmer depth indicates many polymorphic sites in the genome. In fact, using the Genomic Character Estimator program, gce v 1.0.0 [10], the heterozygous ratio based in kmer individuals is 0.054, and the corrected estimate of genome size is 1.32 Gb. Hereon the gce estimate is referred to as the ‘assemblable’ portion of the genome.

A pilot WGS assembly using only PE data was performed in order to generate enough contiguous sequences to gather library insert size statistics. PE reads were first filtered for contaminating sequences (phiX, Escherischia coli and other vector sequences, as well as O. europaea plastids) using GEM [11] with –m 0.02 (2 % mismatches). Then, the reads were assembled into scaffolds using AbySS v1.3.6 [12] with parameters: −s 600 − S 600–3000 − n 6 − N 10 − k 127 − l 75 − aligner map − q 10. This resulted in an assembly with a total length of 1.94 Gb, and contig and scaffold N50s of 3.7 kb and 3.8 kb, respectively. Library insert sizes were estimated by mapping against this draft assembly. For the WGS PE libraries sequenced on Illumina HiSeq2000 using 2x151 bp reads, the insert size distribution followed a bimodal distribution with a main peak at 725 bp and a smaller peak at 300 bp. Before continuing with the assembly, read pairs belonging to the smaller peak were filtered out, if connecting reads were found overlapping both mates of the pair.

The inflated length (47 % of the assemblable part of the genome) and the poor contiguity obtained for the draft assembly are symptomatic of the expected difficulty in distinguishing divergent alleles of the same locus from true repeats. To address this challenge, the 96 sequenced fosmid pools (3.9x physical coverage of the genome, each pool covering ~4 % of the genome) were assembled using the assembly pipeline shown in Fig. 2 to obtain 96 largely haploid assemblies (simulations of 1,600-clone pools with a genome size of 1.38 Gb show a mean of 2.5 % of sequenced bases to derive from separate overlapping clones, half of which would come from different alleles). Optimal kmer size was 97 for most of the pools. For each pool a base assembly was produced using ABySSv1.3.7 and parameters: −s 300 − S 300–5000 − n 9 − N 15 − k 97 − l 75 − aligner map − q 10. Afterwards, the base assemblies went through several rounds of gapfilling [13], decontamination, consistency checks, and rescaffolding with ABySSv1.3.7. The decontamination step consists of detecting contaminant sequences (phiX, vectors, UniVec, E. coli, plastids) in the intermediate assemblies using blastn and masking any matches with Ns, thus producing gaps in the assembly. As a result of the FP pipeline, 96 individual assemblies were obtained with an average scaffold N50 of 33,786 ± 3,105 bp. The distribution of scaffold sizes follows a bimodal distribution (Fig. 3), suggesting that a large fraction of fosmid clones are fully assembled. Mapping of fosmid ends to the merged assembly (‘FP assembly’, see below) gives an estimate of the clone insert size distribution (mean of 36.7 kb ± SD 4.97 kb) that corresponds well with the right peak of the scaffold sizes.

The 96 fosmid pool assemblies were then merged based on overlaps using in-house OLC-like assembly-merging software called ASM (L. Frias and P. Ribeca, manuscript in preparation; scripts are publicly available at [14]. Two rounds of merging were performed, with intermediate scaffolding and gapfilling steps. In the first round, a minimum overlap of 2,400 bp and high sequence similarity (maximum edit distance of 1.5 %) was used, while in the second round, longer overlaps (4,000 bp) and higher sequence divergence (maximum edit distance of 10 %) were used in order to merge allelic regions. Each round of merging collapses repeats unless higher order information supports a unique path for resolving a repetitive region; this includes both the sequence of the input data (contigs) and scaffolding information (i.e., the order of contigs in scaffolds in the original fosmid pool assemblies). Merging produced an intermediate assembly (named ‘FP assembly’ in Fig. 4) with a scaffold N50 of ~45 kb and a total length of 1.38 Gb. Although this assembly was 4.54 % larger than the assemblable genome size (1.32 Gb), gene completeness according to CEGMA was only 95.97 % complete and 97.58 % partial, suggesting that 2.42–4.03 % of the gene space may have been missed.

To increase the overall completeness of the assembly, all WGS reads that did not map to the FP assembly were selected and used to obtain a complementary assembly using ABySSv1.5.2 with parameters: −s 300 − S 300–5000 − n 10 − N 10 − k 95 − l 75 − aligner map − q 10. This assembly accounts for 60.7 Mbp of sequence, and has an N50 of 1,506 bp for contigs and 2,351 bp for scaffolds. This assembly was then broken into contigs, 50 bp was eroded from the ends of each contig, then contigs smaller than 200 bp were filtered out. Both assemblies were subsequently gathered by joining the WGS contigs with the merged fosmid pool assembly, and scaffolding them with SSPACE 2.0 [15]. To account for read pairs coming from two different alleles in the same genomic region, reads were mapped to the SSPACE input assembly with gem-mapper (settings: m = 0.05 and e = 0.1) and filters were applied to detect unique mappings with no subdominant match. The resulting comprehensive assembly had a scaffold N50 of 303.7 kb and a total length of 1.51 Gb, ~190 Mb above the expected genome length (1.32 Gb). The excess of assembled sequence is likely to be caused by the presence of artificial duplications during the assembly process (i.e., uncollapsed haplotypes that have been resolved in two different contigs). Several strategies were used to refine the assembly and obtain a haploid reference. First, consistency check was applied to remove local misassemblies by mapping short and intermediate libraries (PE720, MP3k and MP5k) to the input assembly: a positive score is assigned to the assembly regions supported by read pairs separated by distances falling within the limits (mean ± 3σ) of the empirical distribution, while a negative score is assigned to regions where read pairs map i) outside of these bounds, ii) in inconsistent orientation, or iii) to different scaffolds. Regions where the sum of these two vectors is negative are removed from the assembly. After applying this consistency check, the resulting assembly had 46,893 consistent contig blocks (compared to 25,042 contigs before the consistency check), giving a total of 1.46 Gb with an N50 of 101 kb. Second, this assembly was collapsed using a minimum overlap of 4 kb and the gem-mapper parameters − e 0.03 and − m 0.02, so only close matches were merged (similar uncollapsed haplotypes, identical assembly artifacts, and near identical repeats). Additionally, in order to avoid spurious joins, tip merging was applied to the alignment graph down to overlaps of 250 bp. Finally, no repeat resolution was applied, but coherent links from input scaffolds were reinserted. Consequently, the assembly length shrunk to ~1.30 Gb, almost matching the assemblable fraction of the genome (1.32 Gb). An additional consistency check was run on the collapsed assembly using the short and intermediate libraries (PE720, MP3k and MP5k), which resulted in breaking the assembly from 64,814 into 72,593 scaffolds, giving a total length of 1.30 Gb with a scaffold N50 of 50 kb. This assembly length is what was expected based on the gce estimate. As a final assembly step, PE reads with high divergence (gem-mapper parameters m = 0.05 and e = 0.08) were mapped to the assembly and rescaffolded with SSPACE 2.0 using parameters k = 3 and a = 0.6. Then, scaffolds shorter than 500 bp were discarded, and the GapFiller program [13] was used to close about 40 % of the assembly gaps. This assembly was labeled ‘Oe3’.

The Oe3 assembly was polished using a mapping-based strategy designed to correct single nucleotide substitution and short insertion–deletion errors. First, one library of paired-end reads (PE725) was aligned using BWA mem (v0.7.7) [16] and variant calling was performed. Selecting only homozygous alternative variants, an alternative FASTA sequence was obtained using GATK (v3.5) FastaAlternateReferenceMaker [17]. After discarding scaffolds shorter than 500 bp, the resulting assembly (Oe5) had a scaffold N50 of 444 kb and a contig N50 of 51 kb. After detecting putative contamination in some scaffolds of the Oe5 assembly, a final decontamination step was performed against yeast, bacteria, arthropod and mitochondrial sequences, combining homology search results obtained by BLAST and, in the case of mitochondrial sequences, regions of high depth (~6000x). In total, 509 scaffolds were deleted from Oe5 and some parts of another 27 scaffolds were removed. The assembly resulting from this step, Oe6, has a scaffold N50 of 443 kb and a contig N50 of 52 kb (Table 2). Oe6 contains 48,419 gaps comprising 53,969,601 sites. The gene completeness of this assembly was estimated using CEGMA [18] and BUSCO (Benchmarking Universal Single-Copy Orthologs) [19]. CEGMA analysis resulted in a gene completeness of 98.79 %, while BUSCO, using a plant-specific database of 956 genes, determined a completeness of 95.6 % of plant genes. A summary of the complete assembly strategy is shown in Fig. 4.

Partial assembly of an olive tree associated fungus: Aureobasisium pullulans

One of the putative sources of non-plant sequence present in the olive samples was considered of interest; it was represented among the fosmid pools and seemed to belong to the fungal genus Aureobasidium, which has been previously associated with olive trees [20]. To assemble a partial sequence of this genome, four fully sequenced Aureobasidium genomes were downloaded from JGI [21

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