Improvement of the banana “Musa acuminata” reference sequence using NGS data and semi-automated bioinformatics methods

Abstract

BackgroundRecent advances in genomics indicate functional significance of a majority of genome sequences and their long range interactions. As a detailed examination of genome organization and function requires very high quality genome sequence, the objective of this study was to improve reference genome assembly of banana (Musa acuminata).ResultsWe have developed a modular bioinformatics pipeline to improve genome sequence assemblies, which can handle various types of data. The pipeline comprises several semi-automated tools. However, unlike classical automated tools that are based on global parameters, the semi-automated tools proposed an expert mode for a user who can decide on suggested improvements through local compromises. The pipeline was used to improve the draft genome sequence of Musa acuminata. Genotyping by sequencing (GBS) of a segregating population and paired-end sequencing were used to detect and correct scaffold misassemblies. Long insert size paired-end reads identified scaffold junctions and fusions missed by automated assembly methods. GBS markers were used to anchor scaffolds to pseudo-molecules with a new bioinformatics approach that avoids the tedious step of marker ordering during genetic map construction. Furthermore, a genome map was constructed and used to assemble scaffolds into super scaffolds. Finally, a consensus gene annotation was projected on the new assembly from two pre-existing annotations. This approach reduced the total Musa scaffold number from 7513 to 1532 (i.e. by 80 %), with an N50 that increased from 1.3 Mb (65 scaffolds) to 3.0 Mb (26 scaffolds). 89.5 % of the assembly was anchored to the 11 Musa chromosomes compared to the previous 70 %. Unknown sites (N) were reduced from 17.3 to 10.0 %.ConclusionThe release of the Musa acuminata reference genome version 2 provides a platform for detailed analysis of banana genome variation, function and evolution. Bioinformatics tools developed in this work can be used to improve genome sequence assemblies in other species.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-2579-4) contains supplementary material, which is available to authorized users.

Genome ancestry mosaics reveal multiple and cryptic contributors to cultivated banana

“…Filtered RNA‐seq reads were aligned to Musa reference genomes comprising the M . acuminata nuclear reference sequence (D'Hont et al , ) version 2 (Martin et al , ), 12 mitochondrial scaffolds and the chloroplast genome sequence (Martin et al , ) using the STAR v2.5 aligner software (Dobin et al , ) with no more than 10 mismatches per paired‐end read, an intron size of between 20 bases and 50 kb and no multiple alignment. The mapping process was performed in three steps: (i) first mapping of all paired reads from all libraries; (ii) identification of all splicing sites; and (iii) second mapping of each library independently using splicing site information.…”

“…Filtered DNA‐seq reads were aligned to Musa reference genomes comprising the M . acuminata nuclear reference sequence version 2 (Martin et al , ), 12 mitochondrial scaffolds and the chloroplast genome sequence (Martin et al , ) using bwa v0.7.15 with the mem algorithm (Li, ). Reads aligning at several positions were removed using samtools v1.3 (Li et al , ).…”

Molecular and cytological characterization of the global Musa germplasm collection provides insights into the treasure of banana diversity

“…Briefly, 19 SSR loci (Lagoda et al 1998;Crouch et al 1998;Hippolyte et al 2010) that are well distributed across ten of the eleven Musa genetic linkage groups (Hippolyte et al 2010) were amplified using a set of M13-tailed specific primers to allow universal labelling with fluorescent dyes. Although new SSR markers are accessible thanks to the Musa genome sequence assembly completion (D'Hont et al 2012;Martin et al 2016), the hitherto used marker set was not enlarged, as the reproducibility and comparability of results gathered until now would then not be assured and a re-start of the whole genotyping effort would be inefficient. The PCR reaction mix contained (in the final volume of 20 ll): 10 ng of template genomic DNA, reaction buffer (consisting of 10 mM Tris-HCl (pH 8), KCl 50 mM, 0.1% Triton-X100 and 1.5 mM MgCl 2 ), 200 lM dNTPs (each), 1 U of Taq polymerase, 8 pmol of the M13-tailed locus specific forward primer, 6 pmol of the fluorescently labeled universal M13 forward primer, 10 pmol of the locus specific reverse primer.…”

Translating the “Banana Genome” to Delineate Stress Resistance, Dwarfing, Parthenocarpy and Mechanisms of Fruit Ripening

“…Functional analysis to unravel regulatory details small-, micro-, and si-RNAs and their role in the stress responses and hormone signaling will provide useful information for translational research in banana. As our molecular understanding advances, biotechnological interventions such as gene discovery, genetic engineering, targeted transformation/regeneration, and progressive bioinformatic tools (Martin et al, 2016) coupled with validated markers (Backiyarani et al, 2013) will accelerate breeding and super-domestication of banana will be a reality soon.…”