A High-Quality PacBio Insect Genome from 5 ng of Input DNABrendan Galvin1, Douglas Shoue2, Lei Zhu1, Christine Lambert1, Primo Baybayan1, Sarah Kingan1, Michelle Vierra1,Jonas Korlach1, Mary Ann McDowell2 & Stephen Richards3
1Pacific Biosciences, Menlo Park, CA; 2University of Notre Dame, Notre Dame, IN; 3University of California Davis, Davis, CA

Abstract
High-quality insect genomes are essential
resources to understand insect biology and tocombat them as disease vectors and agricultural
pests. It is desirable to sequence a single individualfor a reference genome to avoid complications frommultiple alleles during de novo assembly. However,the small body size of many insects poses achallenge for the use of long-read sequencing
technologies which often have high DNA-input
requirements. The previously described PacBio
Low DNA Input Protocol starts with ~100 ng of DNA
and allows for high-quality assemblies of single
mosquitoes among others and represents asignificant step in reducing such requirements.
Here, we describe a new library protocol with afurther 20-fold reduction in the DNA input quantity.
Starting with just 5 ng of high molecular weight
DNA, we describe the successful sequencing andde novo genome assembly of a single male sandfly(Phlebotomus papatasi, the main vector of the Old
World cutaneous leishmaniasis), using HiFi data
generated on the PacBio Sequel II System andassembled with FALCON. The assembly shows ahigh degree of completeness (>97% of BUSCO
genes are complete), contiguity (contig N50 of 1Mb), and sequence accuracy (>98% of BUSCO
genes without frameshift errors). This workflow has
general utility for small-bodied insects and otherplant and animal species for both focused researchstudies or in conjunction with large-scale genome
projects.

Example - Sandfly
Phlebotomus papatasi:• Main vector of Old World
cutaneous leishmaniasis
and Phlebotomus fever

Assembly Results:• Falcon assembler + Purge_dups

Phlebotomus fever distribution (Toscana, Sicilian, Naples serotypes)3• Sufficient DNA for low DNA input
protocol1 could not be obtained froma single individual
Library preparation:• DNA extracted from a single sandfly• Made library starting with 5 ng
using the ultra-low DNA input
protocol, followed by BluePippin
size selection (8-15 kb)
Starting DNA

Motivation & Workflow

Example - Sandfly

Thisassembly

Previous
assembly4

Assembly size

363 Mb364 Mb# of contigs

977

139,199142x lessfragmented

Contig N50

1,012 kb

5.8 kb174x more
contiguous

BUSCO(arthropoda)5analysis(n=1,066)

C:97.2%
C: 87.3%[S: 95.9%, D: 1.3%] [S: 84.3%, D: 3.0%]
F: 0.7%, M: 2.1%
F: 10.4%, M: 2.3%

Conserved geneframeshift error
analysis(BUSCO geneset)

14 frame shift
inducing errors(808 kb alignmentspace)

25 frame shift
inducing errors(708 kb alignmentspace)

Change9.9% more
completegenes

11 less errors(in ~100 kblargeralignmentsspace)

Protocols & Reagents• SMRTbell gDNA
Sample Amplification
Kit & detailed workflow
protocol are available

Library Preparation for DNA-Limited
Samples:• DNA from single individuals muchpreferred for de novo assembly to
avoid complications from >2 alleles

Final library for
HiFi sequencing• Standard SMRT Sequencing
protocols require > 5 μg HMW gDNA
• Low DNA Input Protocol1 reducesrequirement to ~100 ng• For some samples (smallorganisms), even ~100 ng HMW
DNA is not possible

Conclusions

Sequencing Results:• Run stats from one SMRT Cell 8M:

Workflow:
DNA isolation &shearing

Starting with 5 ng gDNA
>10 kb average size
Megaruptor 3 shearing

Total yield:
Number of reads:
Raw average RL:
Mean subRL:

626 Gb
6.4 million
97.6 kb
13.2 kb• Demonstrating efficient HiFi readbased PacBio sequencing from as
low as 5 ng of input DNA• High-quality de novo assembly ofsingle sandfly individual• Kits and protocols available

References & Acknowledgments
Amplification

Library preparation& size selection

PacBio HiFi
sequencing

Proprietary (PCR-based)
SMRTbell gDNA Sample
Amplification Kit

HiFi SMRTbell Libraries
using SMRTbell Express
Template Prep Kit 2.02

Sequel II
System>Q20 HiFi reads:
HiFi yield:
Mean HiFi RL:

3,327,164
35.9 Gb
10.8 kb

Mean HiFi read quality:

Q36 (99.975%)

1Kingan et al. (2019) Genes 10: 62
2https://www.pacb.com/wp-

content/uploads/ProcedureChecklist-Preparing-HiFi-SMRTbellLibraries-using-SMRTbell-ExpressTemplate-Prep-Kit-2.0.pdf
3https://commons.wikimedia.org/w/ind
ex.php?curid=5576847
4https://www.ebi.ac.uk/ena/data/view/
GCA_000262795.1
5https://busco.ezlab.org/

The authors would like to thank everyone who helped
generate data for the poster.

For Research Use Only. Not for use in diagnostic procedures. © Copyright 2020 by Pacific Biosciences of California, Inc. All rights reserved. Pacific Biosciences, the Pacific Biosciences logo, PacBio, SMRT, SMRTbell, Iso-Seq, and Sequel are trademarks of Pacific Biosciences.
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