5.7 The Present
At present, there are many strategies that one could employ to perform de novo genome
sequencing of a plant genome. Currently, we would recommend a combination of
Illumina and third-generation sequencing along with Hi-C and/or optical mapping support. At the Institute for Genomics, Biocomputing & Biotechnology, we have employed
the following steps to assemble a number of plant, vertebrate, and insect genomes:
• Sequence 30-genome equivalent (30X) coverage using a third-generation
sequencer. We have utilized both PacBio Sequel and the Oxford Nanopore
MinIon/GridIon instruments (the former through contracts and the latter
“in-house”).
• Assemble the sequence reads into contigs and scaffolds using a Minimap2Minasm-Racon (see Li 2016; Vaser et al. 2017) pipeline.
• Acquire 100X coverage of the genome using a high-throughput Illumina instrument. At present we are using a NovaSeq 6000, a HiSeq X Ten, or a HiSeq 4000.
• Use Illumina reads to polish the third-generation contigs/scaffolds using Pilon
(Walker et al. 2014).
• Use Hi-C to improve quality and length of contigs and scaffolds using the
3D-DNA program (Dudchenko et al. 2017).
• Perform structural annotation of the genome with MAKER2 (Holt and Yandell
2011) using evidence from GeneMark, Augustus, BUSCO, Snap, BLAST+, and
Exonerate (Isono et al. 1994; Stanke et al. 2004; Simao et al. 2015; Zaharia et al.
2011; Camacho et al. 2009; Slater and Birney 2005).
• Conduct functional annotation of the genome with InterProScan and BLAST+
(Zdobnov and Apweiler 2001; Camacho et al. 2009).
6 The Best Plant Genome Sequences
6.1 A Different Perspective
In 2013, Michael and Jackson published a paper entitled “The 50 first plant
genomes.” As its title suggests, this excellent review focused on the 50 first plant
genome publications. The relative quality of the 50 first genomes varies enormously
(e.g., the listed contig N50 for Brachypodium distachyon is 348 kb, while the contig
N50 for Azadirachta indica is 1 kb).
To provide a different perspective, we have chosen to focus not on chronological
publication/release dates but on the highest-quality plant genome assemblies currently available. Some of these genomes have not been described in publication
form, but all are available through the National Center for Biotechnology Information (NCBI) Assembly Database (https://www.ncbi.nlm.nih.gov/assembly/). Table 3
provides a list of those plant genomes that meet the criteria for reference-quality
Sequencing Plant Genomes
165
At present, there are many strategies that one could employ to perform de novo genome
sequencing of a plant genome. Currently, we would recommend a combination of
Illumina and third-generation sequencing along with Hi-C and/or optical mapping support. At the Institute for Genomics, Biocomputing & Biotechnology, we have employed
the following steps to assemble a number of plant, vertebrate, and insect genomes:
• Sequence 30-genome equivalent (30X) coverage using a third-generation
sequencer. We have utilized both PacBio Sequel and the Oxford Nanopore
MinIon/GridIon instruments (the former through contracts and the latter
“in-house”).
• Assemble the sequence reads into contigs and scaffolds using a Minimap2Minasm-Racon (see Li 2016; Vaser et al. 2017) pipeline.
• Acquire 100X coverage of the genome using a high-throughput Illumina instrument. At present we are using a NovaSeq 6000, a HiSeq X Ten, or a HiSeq 4000.
• Use Illumina reads to polish the third-generation contigs/scaffolds using Pilon
(Walker et al. 2014).
• Use Hi-C to improve quality and length of contigs and scaffolds using the
3D-DNA program (Dudchenko et al. 2017).
• Perform structural annotation of the genome with MAKER2 (Holt and Yandell
2011) using evidence from GeneMark, Augustus, BUSCO, Snap, BLAST+, and
Exonerate (Isono et al. 1994; Stanke et al. 2004; Simao et al. 2015; Zaharia et al.
2011; Camacho et al. 2009; Slater and Birney 2005).
• Conduct functional annotation of the genome with InterProScan and BLAST+
(Zdobnov and Apweiler 2001; Camacho et al. 2009).
6 The Best Plant Genome Sequences
6.1 A Different Perspective
In 2013, Michael and Jackson published a paper entitled “The 50 first plant
genomes.” As its title suggests, this excellent review focused on the 50 first plant
genome publications. The relative quality of the 50 first genomes varies enormously
(e.g., the listed contig N50 for Brachypodium distachyon is 348 kb, while the contig
N50 for Azadirachta indica is 1 kb).
To provide a different perspective, we have chosen to focus not on chronological
publication/release dates but on the highest-quality plant genome assemblies currently available. Some of these genomes have not been described in publication
form, but all are available through the National Center for Biotechnology Information (NCBI) Assembly Database (https://www.ncbi.nlm.nih.gov/assembly/). Table 3
provides a list of those plant genomes that meet the criteria for reference-quality
Sequencing Plant Genomes
165