Every species can be a model: Reference-quality
PacBio genomes from single insects
Sarah B Kingan1, Christine C Lambert1, Liuqi Gu2, Katharina von Wyschetzki3, PrimoBaybayan1, Konrad Lohse4, James R Walters2, Mara KL Lawniczak3, Jonas Korlach1
1. PacBio, Menlo Park, CA, 2. University of Kansas, Lawrence, 3. Wellcome Sanger Institute, Hinxton UK,
4. Edinburgh University, UK.

Monarch (Danaus plexippus)

Challenges
Small body size limits amount of genomic
DNA from a single individualHigh heterozygosity samples require diploidaware de novo assembly and curation
strategies
Pooling multiple individuals complicatesbioinformatics analysis
Solutions
PacBio libraries generated from single
insects enabled by Low DNA Input Protocol
Two samples (genome <600 Mb) can be
multiplexed on one SMRT Cell 8M on Sequel
II System
Genome assembly with HiFi reads iscomputationally efficient
Assemblies from HiFi reads are more
complete than other technologies-- Single Female pupa collected- DNA extraction with OmniPrep-Prep with 16 µg DNA

Identifying Sex Chromosomes (ZW)
♂ZZ

♀ZW- Neo-Z chrom resulted from fusion

Kit- Standard HiFi Library
Mongue et al. 2017

HiFi Yield
Q20: 32 Gb400(Thousands)-Paternal Contigs

Read Count

Dapl v3 Z Chrom

30020010005000

10000

15000

20000

HiFi Read Length (Q30)

420050100

150200log2(M/F)

Contig Length (Mbp)-6Neo- and Anc-Z with 7 contigs (left)- Candidate W contigs identified byIllumina read depth analysis2 (below)210−1−2−3−40.5 Mb contig with female-based read mapping

Length (Mb): 250/258
N Contigs = 233/316
Contig N50 (Mb): 3.9/4.4
BUSCO Complete (%): 98/94
BUSCO Duplicate (%): 9/11− maternal assembly− paternal assembly

8- Paternal assembly spans the 16Mb

Sex-Ratio of Illumina Read Depth
for Maternal Assembly- Trio binning + FALCON
10of Ancestral Z and autosome1- W chromosome absent fromprevious reference but cytogenetic
analysis consistent with fused Neoand Anc-W

0−2−4−6−8−10−12250

Log2(M/F)

Insect Assembly

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00.1

Cummulative Assembly Length (Mbp)

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Candidate W(female-biased read depth)

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0.1 0.2
0.5 12
5Contig Length (Mb)

000085F Coordinate (Mbp)

10Red Admiral (Vanessa atalanta)
PacBio Workflows- Single-contig chromosomes with resolved telomere- HMW DNA (>40 kb fragments)- Extraction methods vary by

sequences revealed by alignment to H. melpomene- Single Female collected- DNA extraction with “10Xsample

Heliconius melpomene (Heml2.5)
11.3 kb

modified” protocol3© A.Savin, WikiCommons

ACCTAACCTAACCTAACCTA

Vanessa atalanta

CCTACCTACCTACCTACCTA- Low DNA Input Library
Input DNA Genome Size
15 µg

no limit

Low DNA Input (1-plex)
Low DNA Input (2-plex)

400 ng
300 ng<1 Gb<600 Mb

TAGGTAGGTAGGTAGGTAGG

S150(Thousands)

Standard HiFi Library

Read Count

Library
ProtocolPrep17.1 kb
Contig 000010F
TTAGGTTAGGTTAGGTTAGG

Known telomere repeat3

HiFi Yield
Q20: 13 Gb

10050005000

10000 15000 20000- Diversity in

HiFi Read Length (Q20)

HiFi Reads- Accuracy: >99%
- Length: 5-25 kb- Coverage: 15 to 20fold

telomerestructureContig Length (Mbp)- FALCON + Purge-Dups
15Length: 368 Mb
N Contigs = 80
Contig N50: 10.13 Mb
BUSCO Complete: 99.1%
BUSCO Duplicate: 0.3%10500100

200300Contig 000016F

Contig 000003F

Contig 000001F

Contig 000002F

Contig 000029F

Contig 000013F

Cummulative Assembly Length (Mbp)

Estimate genomesize, heterozygositywith Genomescopeusing HiFi reads- Single Females collected- DNA extraction with “10Xmodified” protocol3

Jim Gathany, Wikipedia

or Purge Haplotigs- Low DNA Input Prep (multiplex2 samples)- 230 ng input DNA per sample
barcodeAssembly is fast with HiFi reads- Sample 1 subreads assembled and compared to HiFi assembly
Sample 1HiFi Read AssemblyLong Read Assembly

Coverage

25-fold

40-fold

N50 Read Length (N5)

11 kb (19 kb)

12 kb (22 kb)

Primary Asm Length262 Mb243 MbPrimary Contig N50

5.28 Mb

3.86 Mb465

212

C:98.7%, D:0.1%
F:0.6%, M:0.7%

C:98.7, D:0.2%
F:0.6%, M:0.7%

1604

1947

Primary Contigs
BUSCO
CPU Hours (Consensus + Assembly)

HiFi Yield (Gb)Sample 1: 6.9
Sample 2: 6.3

25020015010050
0

HiFi assemblies capture satellites and other repeats
05000

15000

25000

HiFi Read Length (Q20)

Contig Length (Mbp)

All protocols can be found on our website:- www.pacb.com/documentation/
BUSCO: Waterhouse RM et al. (2017) Mol Biol Evol, 35(3):543–548
Purge Dups: https://github.com/dfguan/purge_dups
FALCON: https://github.com/PacificBiosciences/pb-assembly
Canu: https://github.com/marbl/canu
Purge haplotigs: https://bitbucket.org/mroachawri/purge_haplotigs/
Racon: https://github.com/lbcb-sci/racon
Genomescope: https://github.com/schatzlab/genomescope
1. Mongue A. J. et al. (2017) G3: 7:3281
2. Killick R., and Eckley, I. (2014). J of Stat Software, 58:1t
3. Kingan S., et al. (2019) Genes, 10:62
4. Vitková M., et al. (2005) Chrom Res. 13:145
The authors wish to thank Michelle Vierra, Greg Concepcion, MaggieWeitzman, Kristin Mars, Nick Sisneros, Adam Knight, Kristin Robertshaw,Pamela Bentley Mills

Read Count

References and Acknowledgements(Thousands)- FALCON for HiFi reads- Polish with racon- Haplotype deduplication with Purge Dups

Mosquito (Anopheles coluzzii)- FALCON + Purge-Dups- 9 Mb of HiFi Read assembly does not map to Long Read assembly- Primarily map to “UNKN” (96%) or sex chromosomes (3% Y, 1% X)- A known satellite repeat (AgX367, L = 367 bp) maps across contig (below)

Length (Mb): 262/259
N Contigs = 465/358
Contig N50 (Mb): 5.3/2.9
BUSCO Complete: 98.8/98.8
BUSCO Duplicate: 0.1/0.3

151050050100150200250000169F (L = 140 kb)

Cummulative Assembly Length (Mbp)

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