PacBio library construction and sequencing and filling gaps

The preliminary assembly of A. shenzhenica and the previous published genome assemblies of P. equestris⁹ and D. catenatum¹⁰ were improved using PacBio and 10X Genomics Linked-Reads.

Genomic DNA was isolated from the leaves of A. shenzhenica, P. equestris and D. catenatum. For a 20-kb insert size library, at least 20 μg of sheared DNA was required. SMRTbell template preparation involved DNA concentration, damage repair, end repair, ligation of hairpin adapters, and template purification, and used AMPure PB Magnetic Beads. Finally, the sequencing primer was annealed and sequencing polymerase was bound to SMRTbell template. The instructions specified as calculated by the RS Remote software were followed. We carried out 20-kb single-molecule real-time DNA sequencing by PacBio and sequenced the DNA library on the PacBio RS II platform, yielding about 5.44 Gb (A. shenzhenica), 10.54 Gb (P. equestris) and 11.06 Gb (D. catenatum) PacBio data (read quality ≥ 0.80, mean read length of A. shenzhenica ≥ 7 Kb, of P. equestris and D. catenatum ≥ 10 Kb) (Supplementary Table 2).

We used PBjelly software³⁵ to fill gaps with PacBio data. The options were “<blasr>-minMatch 8 -sdpTupleSize 8 -minPctIdentity 75 -bestn 1 -nCandidates 10 -maxScore -500 -nproc 10 -noSplitSubreads</blasr>” for the protocol.xml file. Then, we used Pilon³⁶ with default settings to correct assembled errors. For the input BAM file, we used BWA to align all the Illumina short reads to the assembly and SAMTOOLS to sort and index the BAM file.