Abstract Detail

Comparative Genomics/Transcriptomics

De La Cerda, Gisel [1], Landis, Jacob [2], Hernandez, Adriana [3], Tribble, Carrie [4], Eifler, Evan [5], Li, Fay-Wei [6], Zhang, Jing [7], Karimi, Nisa [8], Chan, Patricia [9], Givnish, Thomas [10], Strickler, Susan [11], Specht, Chelsea [12].

Balancing read length and sequencing depth: optimizing Nanopore long-read sequencing for monocots with an emphasis on Liliales.

Generating long-read sequencing data for nonmodel plant species can be challenging, especially for species containing secondary metabolites. Many protocols exist for extracting high molecular weight DNA, however additional steps may be necessary to generate clean DNA for Nanopore sequencing. Protocols that work in one taxonomic group are not guaranteed to work in all groups. Here we present approaches that have been used to generate long-read Nanopore sequencing reads for Liliales, as well as other monocot species, and how these modifications directly impact read length and total output for a flow cell. Overall, four species of Calochortus were sequenced with four or five flow cells per species. Modifications to a standard SDS extraction protocol include grinding with a mortar and pestle, using cut/widebore tips, chloroform cleaning, bead cleaning, eliminating short fragments, and generating clean DNA with kits. With slight modifications in protocols among different replicates of the same species we obtain a clear view of the direct impact different modifications have on sequencing output. Notably, maximizing read length can decrease overall output and the number of pores available in a flow cell is not correlated with the overall output. The goal of this paper is to help those interested in generating long-reads determine which steps may be necessary for optimizing long-read sequencing in their taxa of interest, especially within the monocots.

1 - 419 E King Rd , Ithaca, CA, 14850, United States
2 - Cornell University
3 - Cornell University, School of Integrative Plant Science, Section of Plant Biology and the L.H. Bailey Hortorium, Ithaca, NY, 14853, United States
4 - University Of Hawaii At Manoa, Department Of Biology, 2538 McCarthy Mall, Edmondson Hall 216, Honolulu, HI, 96822, United States
5 - UW Madison, Botany, 430 Lincoln Dr., Madison, WI, 53706, United States
6 - Boyce Thompson Institute, 105 Valley Road, Ithaca, NY, 14850, United States
7 - Boyce Thompson Institute
8 - University Of Wisconsin - Madison, Department Of Botany, 430 Lincoln Drive, Madison, WI, 53706, United States
9 - University Of Wisconsin Madison, Botany, Birge Hall, Madison, WI, 53703, United States
10 - University Of Wisconsin-Madison, Department Of Botany, 315 Birge Hall, 430 Lincoln Drive, Madison, WI, 53706, United States
11 - Boyce Thompson Institute For Plant Research , 533 Tower Road, Ithaca, NY, 14853, United States
12 - Cornell University, Plant Biology, Plant Science, 236 Tower Road, Ithaca, NY, 14851, United States

Keywords:
Nanopore
genome sequencing
Calochortus
monocot.

Presentation Type: Oral Paper
Number: CGT3001
Abstract ID:179
Candidate for Awards:None