Spotlight: Plant Genomes

Easily isolate long DNA fragments that overcome challenges of sequencing large and complex plant genomes

The problem with plant genomes

Plant genomes tend to be large, repetitive, polyploid, heterozygous, and highly plastic. Enrichment and genome sequencing methods used to date have proven inadequate or just expensive.

  • Genome drafts are often fragmented and include low-quality assemblies1

  • WGS is inefficient and expensive to investigate specific genes or regions

  • Widely used long-range PCR followed by Sanger sequencing is limited in length (~10 kb) and requires knowledge of the target sequence

  • Genome walking or using BACs to target sequencing is laborious and difficult to design

Plant genomes in perspective

Plant Genome 01

Angiosperm genome sizes span 4 orders of magnitude2

Plant Genome 05

Percent of genome consisting of repeat sequences and transposable elements increases with genome size2

Plant Genome 03

Most published plant genomes are 15 % incomplete due to high copy number repeats2

How does Xdrop™ help?

 

FEATURES OF Xdrop™ TECHNOLOGY

APPLICATIONS THAT BENEFIT FROM Xdrop™ TECHNOLOGY

Work with unknown
sequences
Long-range genomic
information (100 kb)
Single-molecule
resolution
Easy design &
workflow
Analyze regions of genomes that are poorly characterized or for which there is only a distant reference sequence
Close gaps due to low-quality genome assemblies or low correspondence between reference genome and plant variety
Resolve structural rearrangements (InDels, transposable elements), tandem repeats, duplications, and pseudogenes
Uncover sequence variation between duplicated genes or among copies of a gene family and understand their functional significance
Examine biosynthetic gene clusters based on candidate genes without performing WGS, PCR amplification, or Sanger sequencing
Expand QTL mapping or bulked segregant analysis (BSA) to identify completely linked trait markers and causative polymorphisms
Capture long stretches of DNA from polyploid genomes for unbiased deep sequencing to phase haplotypes
Simplify sample preparation for long-read sequencing to tackle whole genome sequencing of large and complex plant genomes

APPLICATIONS THAT BENEFIT FROM Xdrop™ TECHNOLOGY

Analyze regions of genomes that are poorly characterized or for which there is only a distant reference sequence:
  • Long-range genomic information (100 kb)
  • Easy design & workflow
Close gaps due to low-quality genome assemblies or low correspondence between reference genome and plant variety:
  • Work with unknown sequences
  • Long-range genomic information (100 kb)
  • Single-molecule resolution
  • Easy design & workflow
Resolve structural rearrangements (InDels, transposable elements), tandem repeats, duplications, and pseudogenes
  • Work with unknown sequences
  • Long-range genomic information (100 kb)
  • Single-molecule resolution
  • Easy design & workflow
Uncover sequence variation between duplicated genes or among copies of a gene family and understand their functional significance
  • Long-range genomic information (100 kb)
  • Single-molecule resolution
Examine biosynthetic gene clusters based on candidate genes without performing WGS, PCR amplification, or Sanger sequencing
  • Work with unknown sequences
  • Long-range genomic information (100 kb)
  • Easy design & workflow
Expand QTL mapping or bulked segregant analysis (BSA) to identify completely linked trait markers and causative polymorphisms
  • Work with unknown sequences
  • Long-range genomic information (100 kb)
  • Easy design & workflow
Capture long stretches of DNA from polyploid genomes for unbiased deep sequencing to phase haplotypes
  • Long-range genomic information (100 kb)
  • Single-molecule resolution
Simplify sample preparation for long-read sequencing to tackle whole genome sequencing of large and complex plant genomes
  • Long-range genomic information (100 kb)
  • Single-molecule resolution
  • Easy design & workflow

Xdrop™ saves time and resources

Much less sequencing for far greater insights.

We used Xdrop™ to investigate the gene cluster that synthesizes falcarindiol in the tomato plant. Metabolic and mRNA expression analyses published by researchers at Stanford University had identified 8 candidate genes of the cluster.3 Alas, only 2 were partially matched in the reference genome and an attempted WGS using Oxford Nanopore Technology generated 4 Gb of data but only 3 reads in the region of interest! With Xdrop™, we achieved 977-fold enrichment of a 100 kb region around one of the identified genes. Sequencing coverage when reads were mapped was about 80x in the target region, and some reads even spanned missing genes in the reference genome (visible as breakpoints in the coverage graph).

tomato cov

Sources

1 Schatz, M.C., et al. 2012. Current challenges in de novo plant genome sequencing and assembly. Genome Biol. 13: 243.
2 Michael, T.P. 2014. Plant genome size variation: bloating and purging DNA. Brief. Funct. Genomics 13: 308.
3 Jeon, J.E., et al. 2020. A pathogen-responsive gene cluster for highly modified fatty acids in tomato. Cell 180: 176.

How does Xdrop™ work?

With a simple workflow, Xdrop™ enriches large DNA fragments based on knowledge of only a small sequence in or flanking a region of interest. Those enriched fragments are then individually amplified to create a sequencing library that is truly representative of your sample and which can be used in both long- and short-read sequencing to elucidate the gene content of complex plant genomes.