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Long-read, whole-genome shotgun
sequence data for five model
organisms
Kristi E. Kim
1
, Paul Peluso
1
, Primo Babayan
1
, P. Jane Yeadon
2
, Charles Yu
3
,
William W. Fisher
3
, Chen-Shan Chin
1
, Nicole A. Rapicavoli
1
, David R. Rank
1
, Joachim Li
4
,
David E.A. Catcheside
2
, Susan E. Celniker
3
, Adam M. Phillippy
5
, Casey M. Bergman
6
& Jane M. Landolin
1
Single molecule, real-time (SMRT) sequencing from Pacific Biosciences is increasingly used in many areas of
biological research including de novo genome assembly, structural-variant identification, haplotype phasing,
mRNA isoform discovery, and base-modification analyses. High-quality, public datasets of SMRT sequences
can spur development of analytic tools that can accommodate unique characteristics of SMRT data (long
read lengths, lack of GC or amplification bias, and a random error profile leading to high consensus
accuracy). In this paper, we describe eight high-coverage SMRT sequence datasets from five organisms
(Escherichia coli,Saccharomyces cerevisiae,Neurospora crassa,Arabidopsis thaliana, and Drosophila
melanogaster) that have been publicly released to the general scientific community (NCBI Sequence Read
Archive ID SRP040522). Data were generated using two sequencing chemistries (P4C2 and P5C3) on the
PacBio RS II instrument. The datasets reported here can be used without restriction by the research
community to generate whole-genome assemblies, test new algorithms, investigate genome structure and
evolution, and identify base modifications in some of the most widely-studied model systems in biological
research.
Design Type(s) observation design •genome sequencing •Shotgun Sequencing
Measurement Type(s) DNA sequencing
Technology Type(s) PacBio RS II
Factor Type(s)
Sample Characteristic(s)
Escherichia coli str. K-12 substr. MG1655 •Saccharomyces cerevisiae W303 •
Neurospora crassa OR74A •Neurospora crassa •Arabidopsis thaliana •
Drosophila melanogaster
1
Pacific Biosciences of California Inc., 1380 Willow Road, Menlo Park, California 94025, USA.
2
Flinders University,
School of Biological Sciences, PO Box 2100, Adelaide, South Australia 5001, Australia.
3
Department of Genome
Dynamics, Lawrence Berkeley National Laboratory, 1 Cyclotron Road, Berkeley, California 94720, USA.
4
Department of Microbiology and Immunology, UCSF, San Francisco, California 94158, USA.
5
National
Biodefense Analysis and Countermeasures Center, 110 Thomas Johnson Drive, Frederick, Maryland 21702,
USA.
6
Faculty of Life Sciences, University of Manchester, Oxford Road, Manchester M13 9PT, UK.
Correspondence and requests for materials should be addressed to J.M.L. (email: jlandolin@pacificbiosciences.com)
OPEN
SUBJECT CATEGORIES
» Genomics
» Genome assembly
algorithms
» DNA sequencing
» Experimental organisms
Received: 08 August 2014
Accepted: 03 October 2014
Published: 25 November 2014
www.nature.com/scientificdata
SCIENTIFIC DATA |1:140045 |DOI: 10.1038/sdata.2014.45 1
Background & Summary
Single-molecule, real-time (SMRT®) DNA sequencing occurs by optically detecting a fluorescent signal
when a nucleotide is being incorporated by a DNA polymerase
1
. This relatively new technology enables
detection of DNA sequences that have unique characteristics, such as long read lengths, lack of CG bias,
random error profiles, and can yield highly accurate consensus sequences. Kinetic information such as
pulse width and interpulse duration are also recorded and can be used to detect base modifications
2,3
.
Since its introduction, investigators have published on a range of applications using SMRT sequencing.
For example, the developers of GATK (Genome Analysis Toolkit) demonstrated that single nucleotide
polymorphisms (SNPs) could be detected using SMRT sequences
4,5
due to their lack of context-specific
bias and systematic error
5,6
. Likewise, the developers of PBcR (PacBio error correction)
7,8
showed that
complete bacterial genome assemblies using SMRT sequence data were achievable and had greater than
Q60 consensus base quality
8
. PBcR was later incorporated as the ‘pre-assembly’step in the HGAP
(hierarchical genome assembly process) system
9
, followed by consensus polishing using the Quiver
algorithm
9
to produce a complete assembly pipeline in SMRT Analysis, a free and open-source software
suite released by Pacific Biosciences. In addition, other third-party tools now support long reads for
various applications such as mapping
10,11
, scaffolding
12
, structural-variation discovery
13
, and genome
assembly
7,14
. Other applications such as 16S rRNA sequencing
15
, characterization of entire
transcriptomes in chickens
16
and humans
17
, genome-editing studies
18
, base-modification studies
19–22
,
and validation of CRISPR targets
23
have also been published. Several datasets from this publication have
already been used to develop the MinHash Alignment Process (MHAP), a new method for fast and
efficient overlap of long reads for assembling large genomes
24
.
To encourage interest in further applications and tool development for SMRT sequence data, we
report here the release of eight whole-genome shotgun-sequence datasets from five model organisms
(E. coli,S. cerevisiae,N. crassa,A. thaliana, and D. melanogaster). These organisms have among the most
complete and well-annotated reference genome sequences, due to continual refinement by dedicated
teams of scientists. Despite continued improvement of these genome sequences with new technologies,
few are completely finished with fully contiguous assemblies of all chromosomes. The gaps remaining
arise from complex structures such as transposable elements, repeats, segmental duplications, or other
dynamic regions of the genome that cannot be easily assembled. Structural differences in these regions
can account for variability in millions of nucleotides within every genome, and mounting evidence
suggest that such mutations are important for human diversity and disease susceptibility in many
complex traits
25
including autism and schizophrenia
26
. SMRT sequencing data can therefore play an
important role in the completion of these and other reference genomes, providing a platform for new
insights into genome biology.
Methods
We generated eight whole-genome shotgun-sequence datasets from five model organisms using the P4C2
or P5C3 polymerase and chemistry combinations, totaling nearly 1000 gigabytes (GB) of raw data (See
Data Records section). Genomic DNA was either purchased from commercial sources or generously
provided by collaborators.
Genomic DNA sample summaries are provided in Table 1. DNA from the reference K12 strain of
E. coli was purchased from Lofstrand Labs Limited (K12 MG1655 E. coli, cat# L3-4001SP2). DNA from
the reference OR74A strain of N. crassa was purchased from the Fungal Genetics Stock Center (FGSC).
A standard Ler-0 strain of A. thaliana plants was grown from seeds purchased from Lehle seeds (WT-04-
19-02) and DNA was extracted at Pacific Biosciences. Theprotocol is available on Sample Net
27
and
Dataset Name Sample ID DNA extraction gDNA size
(kb)
Shearing Size selection
E. coli MG1655 P4C2 SAMN02951645 ammonium acetate or SDS, proteinase K, phenol-chloroform 17 none Blue Pippin (7 kb)
E. coli MG1655 P5C3 SAMN02743420 ammonium acetate or SDS, proteinase K, phenol-chloroform 17 none Blue Pippin (7 kb)
S. cerevisiae 9464 P4C2 SAMN02731377 Qiagen genomic DNA buffer set 40 g-TUBE Blue Pippin (17 kb)
N. crassa OR74A P4C2 SAMN02724975 BashingBeads, Zymo Research kit 6 none Blue Pippin (4 kb)
N. crassa T1 P4C2 SAMN02724976 SDS, proteinase K, phenol-chloroform, RNAase, isopropanol 15 none Blue Pippin (7 kb)
A. thaliana Ler-0 P4C2 SAMN02731378 CTAB, chloroform:isoamyl, isopropanol precip. 40 g-TUBE Blue Pippin (7 kb)
A. thaliana Ler-0 P5C3 SAMN02724977 CTAB, chloroform:isoamyl, isopropanol precip. 40 g-TUBE Blue Pippin (15 kb)
D. melanogaster ISO1
P5C3
SAMN02614627 SDS, phenol-chloroform, CsCl banding, ethanol precip. 40 g-TUBE Blue Pippin (17 kb)
Table 1. Summary of DNA samples. The NCBI sample ID associated with each dataset is provided. DNA
was extracted in a species-specific manner, yielding genomic DNA of various sizes. All DNA was size
selected using the Blue Pippin system (Sage Sciences), and select samples were sheared with g-TUBEs
(Covaris).
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SCIENTIFIC DATA |1:140045 |DOI: 10.1038/sdata.2014.45 2
summarized in the organism-specific methods section of this paper. DNA from the 9464 strain of
S. cerevisiae was provided by J. Li at University of California San Francisco. The 9464 strain is a daughter
of the reference WG303 strain. DNA from the T1 strain of N. crassa was obtained from D. Catcheside at
Flinders University who has an interest in polymorphic genes regulating recombination. The T1 strain is
an A mating type strain which, like OR74A, was derived from a cross between the Em a 5297 and Em A
5256 strains. DNA from the ISO1 strain
28
of D. melanogaster was obtained from S. Celniker at Lawrence
Berkeley National Laboratory. This is the reference strain of D. melanogaster that was originally chosen to
be the first large genome to be sequenced and assembled using a whole-genome shotgun approach
29
.It
continues to serve as the reference strain in subsequent releases and numerous annotations of the
D. melanogaster genome.
DNA extraction methods were species-specific and optimized for each organism (see organism-
specific methods below). In general, the steps are: (1) remove debris and particulate material, (2) lyse
cells, (3) remove membrane lipids, proteins and RNA, (4) DNA purification.
SMRTbell™libraries for sequencing
4
were prepared using either 10 kb
30,31
or 20 kb
32
preparation
protocols to optimize for the most high-quality and longest reads. The main steps for library preparation
are: (1) shearing, (2) DNA damage repair, (3) blunt end-ligation with hairpin adapters supplied in the
DNA Template Prep Kit 2.0 (Pacific Biosciences), (4) size selection, and (5) binding to polymerase using
the DNA Sequencing Kit 3.0 (Pacific Biosciences).
E. coli collection, DNA extraction, and SMRTbell library preparation
Both P4C2 and P5C3 samples were prepared in the same way. E. coli K12 genomic DNA was ordered and
purified by Lofstrand Labs Limited (K12MG1655 E. coli, cat# L3-4001SP2). Field Inversion Gel
Electrophoresis (FIGE) was run to ensure presence of high-molecular-weight gDNA. Ten micrograms of
gDNA was sheared using g-TUBE devices (Covaris, Inc) spun at 5,500 r.p.m. or 2029 g on the MiniSpin
Plus (Eppendorf) for 1 min. Three microliters of elution buffer (EB) was added to rinse the upper
chamber, spun at 6,000 r.p.m., or 2415 g and spun again at 5,500 r.p.m. or 2029 g on the MiniSpin Plus
(Eppendorf) after inverting the g-TUBE device. SMRTbell libraries were created using the ‘Procedure &
Checklist—20 kb Template Preparation using BluePippin™Size Selection’protocol
32
. Briefly, the library
was run on a BluePippin system (Sage Science, Inc., Beverly, MA, USA) to select for SMRTbell templates
greater than 10 kb. The resulting average insert size was 17 kb based on 2100 Bioanalyzer instrument
(Agilent Technologies Genomics, Santa Clara, CA, USA). Sequencing primers were annealed to the
hairpins of the SMRTbell templates followed by binding with the P5 sequencing polymerase and
MagBeads (Pacific Biosciences, Menlo Park, CA, USA). One SMRT Cell was run on the PacBio®
RS II system with an on-plate concentration of 150 pM using P5C3 chemistry and a 180-minute
data-collection mode.
S. cerevisiae collection, DNA extraction, and SMRTbell library preparation
The 9464 strain is a MAT a haploid strain derived from a w303 reference strain following three
integration events: 1) a construct (pTef2-dTomato-kanCMX6) was inserted near the CEN4 gene; 2) a
construct (pTEF2-eGFP, natMX) was inserted near the ESP gene; and 3) the pds1 gene was deleted and
replaced with the URA3 gene from Kluyveromyces lactis. Cells were grown to an OD600 of ~2 and 350
OD units of cells corresponding to roughly 7 × 10
9
cells were harvested by centrifugation. Cells were
washed in 4 ml of TE then resuspended in 4 ml of Buffer Y1 (Qiagen genomic DNA prep) and
spheroplasted by addition of 250 units of Zymolyase 100T (Seikagaku 120493) for 40 min at 30 °C.
Spheroplasts were pelleted and re-suspended in 5 ml of Qiagen Buffer G2 containing 300 micrograms of
Qiagen RNAse to lyse the cells. 2 mg of proteinase K was then added to the lysate, which was incubated at
50 °C for 30 min. The lysate was then centrifuged at 5000 G for 10 min at 4 °C, and the supernatant was
purified on a Qiagen 100/G genomic prep tip as per Qiagen instructions. The eluted DNA was spooled by
addition of 3.5 ml of isopropanol to the 5 ml of eluated. The DNA was washed in 70% EtOH, air dried,
and re-suspended in 200 microliters of TE by slowly dissolving overnight at room temperature. SMRTbell
libraries were created using the ‘Procedure and Checklist–10 kb Template Preparation and Sequencing
(with Low-Input DNA)’protocol
30
. Twelve SMRT Cells were run on the PacBio RS II system using P4C2
chemistry and a 180-minute data collection mode.
N. crassa OR74A, collection, DNA extraction, and SMRTbell library preparation
N. crassa OR74A was purchased from FGSC# 2489. Cells were inoculated to a density of 4× 10
6
conidia
in 75 ml Vogel’s minimal medium (Medium N)
33
and incubated at room temperature with gentle shaking
for approximately 48 h. Visual inspection shows the culture prior to harvest and demonstrates that there
was only vegetative tissue and no asexual sporulation or induction of aerial hyphae. Mycelia was blotted
dried on sterile paper toweling and pulverized for approximately 30 s at half of the maximum setting in a
Biospec Products Mini BeadBeater tissue disruptor using the disrupting beads provided with the Zymo
Research ZR fungal/bacterial DNA midi prep kit. Tissue was removed with a sterile inoculating stick and
100 mg of dried mycelia per sample was processed according to the manufactures instructions. DNA was
eluted into 100 ul sterile water and DNA from two samples was pooled. Yield was quantified using a nano
drop system and also validated by agarose gel electrophoresis. The concentration was 32.57 ng/ul, A260
was 0.651, A280 was 0.373, 260/280 was 1.75 and 260/230 was 0.91. The genomic DNA was
www.nature.com/sdata/
SCIENTIFIC DATA |1:140045 |DOI: 10.1038/sdata.2014.45 3
approximately 6 kb and was not sheared. SMRTbell libraries were created using the ‘Procedure and
Checklist—10 kb Template Preparation and Sequencing (with Low-Input DNA)’protocol
30
. Two SMRT
Cells were run on the PacBio RS II system using P4C2 chemistry and a 180-minute data collection mode.
N. crassa T1 collection, DNA extraction, and SMRTbell library preparation
The T1 strain of N. crassa, is an A mating type strain derived by DG Catcheside from a cross between the
Em a 5297 and Em A 5256 strains he obtained from Stirling Emerson in 1955. The fungus was grown in
shake culture for 72 h at 25 °C in 500 ml Vogel’sN
34
minimal medium containing 2% sucrose. Mycelium
was harvested by filtration, ground in liquid nitrogen, resuspended in 10 ml of a buffer containing 0.15 M
NaCl, 0.1 M EDTA, 2% SDS at pH 9.5, and incubated overnight at 37 °C with 1 mg protease K. Debris
was precipitated by centrifugation and 10 ml distilled water was added to the supernatant, which was
extracted once with an equal volume of water-saturated phenol and once with chloroform. Nucleic acids
were precipitated from the aqueous phase with 0.6 volumes of isopropanol. Following centrifugation, the
pellet was dried and dissolved in 1 ml TE buffer (TRIS 10 mM, 1 mM EDTA pH 8.0). RNA and protein
were digested by overnight incubation at 37 °C with RNAase (50 μg) followed by addition of protease K
(50 μg) and further incubation for 2 h. The digest was extracted once with water-saturated phenol and
once with chloroform. DNA was collected by precipitation with 0.6 volumes of isopropanol and,
following centrifugation, the pellet was dried, dissolved in 500 μl TE buffer and stored at 4 °C. Field
Inversion Gel Electrophoresis (FIGE) was run to ensure presence of high-molecular-weight gDNA. The
genomic DNA was approximately 15 kb and was not sheared. SMRTbell libraries were created using the
‘Procedure and Checklist—10 kb Template Preparation and Sequencing (with Low-Input DNA)’
protocol
30
. Eighteen SMRT Cells were run on the PacBio RS II system using P4C2 chemistry and a
180-minute data-collection mode.
A. thaliana collection, DNA extraction, and SMRTbell library preparation
Plants were grown from seeds provided by Lehle seeds (WT-04-19-02). Shoots and leaves were harvested
at three weeks and ground in liquid nitrogen using a mortar and pestle. The complete protocol is
described in the ‘Preparing Arabidopsis Genomic DNA for Size-Selected ~20 kb SMRTbell™Libraries’
protocol
35
. This protocol can be used to prepare purified Arabidopsis genomic DNA for size-selected
SMRTbell templates with average insert sizes of 10–20 kb. We recommend starting with 20–40 grams of
three-week-old Arabidopsis whole plants, which can generate >100 μg of purified genomic DNA.
SMRTbell libraries were created using the ‘Procedure & Checklist—20 kb Template Preparation using
BluePippin™Size Selection’protocol
32
. Eighty-five SMRT Cells were run on the PacBio RS II system using
P4C2 chemistry and a 180-minute data-collection mode. Forty-six SMRT Cells were run on the PacBio
RS II system using P5C3 chemistry and a 180-minute data-collection mode.
D. melanogaster collection, DNA extraction, and SMRTbell library preparation
A total of 1.2 g of adult male ISO1 flies corresponding to 1950 animals were collected, starved for 90–120
min and frozen. The flies ranged in age from 0–7 days based on four collections (1) 0–2 days old, 500
males, 0.33 g; (2) 0–4 days old, 500 males, 0.29 g; (3) 0–7 days old, 500 males, 0.29 g; (4) 0–2 days old, 450
males, 0.29 g. Flies were ground in liquid nitrogen to a fine powder and genomic DNA was purified by
phenol-chloroform extraction and CsCl banding in the ultracentrifuge. Briefly, the pulverized fly extract
was gently re-suspended in 15 ml of HB buffer (7 M Urea, 2% SDS, 50 mM Tris pH7.5, 10 mM EDTA
and 0.35 M NaCl) and 15 ml of 1:1 phenol/chloroform. The mixture was shaken slowly for 30 min and
then centrifuged at 23,600 g for 10 min at 20 °C in a Sorvall HB-4 rotor. The aqueous phase was
re-extracted twice as above and then precipitated by adding two volumes of ethanol and centrifuging at
23,600 g for 10 min at 20 °C in a Sorvall HB-4 rotor. The pellet was re-suspended in 3 ml of TE (10 mM
Tris 1 mM EDTA pH 8.0) by gentle inversion for 3 h. To the re-suspended DNA, 3 g CsCl and 0.3 ml of
10 mg/ml ethidium bromide (EtBr) were added and the mixture centrifuged at 199,000 g for 16 h at 15 °C
in a Beckman VTi 65.2 rotor. The EtBr was removed by extraction with water-saturated butanol,
performed 3 times in a Beckman JA-12 rotor at 13,000 g for 5 min. at 4 degree C each time. The DNA was
diluted three-fold with TE, 1/10 vol, 4 M NaCl was added and the DNA precipitated with two volumes of
ethanol. Centrifugation was done in a JA-12 rotor at 16,000 g for 30 min at 4 degree C for the
precipitation step. After centrifugation, the pellet was washed in 70% ethanol. The DNA was resuspended
in 100 μl TE at a concentration of 1.4 μg/μl and quantified using a Nanodrop instrument. This protocol
routinely yields at least 10 ng DNA per mg of flies with an estimated DNA size >100 kb.Genomic DNA
was sheared using a g-TUBE device (Covaris) at 4800 r.p.m. or 1545 g on the MiniSpin Plus (Eppendorf),
150 ng/μl and purified using 0.45 × volume ratio of AMPure PB beads. SMRTbell libraries were created
using the Procedure & Checklist—20 kb Template Preparation using BluePippin™Size Selection
32
.
Libraries were ligated with excess adapters and an overnight incubation was performed to increase the
yield of ligated fragments larger than 20 kb. Smaller fragments and adapter dimers were then removed by
>15 kb size selection using the BluePippin DNA size selection system by Sage Science. Forty-two SMRT
Cells were run on the PacBio RS II system. The first run was composed of four SMRT Cells, loaded at 75
pM, 150 pM, 300 pM, and 400 pM in order to determine the optimal loading concentration of the
sample. The remaining 38 SMRT Cells were loaded at 400 pM.
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SCIENTIFIC DATA |1:140045 |DOI: 10.1038/sdata.2014.45 4
Data Records
After DNA extraction, libraries were generated and sequenced at Pacific Biosciences of California,
uploaded to Amazon Web Services' Simple Storage Service (S3), and then submitted to the Sequence Read
Archive (SRA) at NCBI under Project ID SRP040522 (Data Citation 1). The corresponding accession
numbers and file sizes are listed in Table 2. More detailed information including md5 checksums and
links to download the original data from AWS S3 are provided in Supplementary Table 1.
Raw data was transferred from the instrument to a storage location and organized first by the run
name, and then by the SMRT Cell directory. Each run contained one or more SMRT Cells. Each SMRT
Cell produced a metadata.xml file that recorded the run conditions and barcodes of sequencing kits, three
bax.h5 files that contained base call and quality information of actual sequenced data, and one bas.h5 file
that acted as a pointer to consolidate the three bax.h5 files. The ‘h5’suffix denotes that these are
Hierarchical Data format 5 (HDF5) files. The specific contents and structure of a PacBio bax.h5 file is
described in more detail in online documentation
36
.
Recall the ‘SMRT bell’structure that underwent sequencing was created by the library preparation
process
4
. Sequenced SMRT Bells corresponded to raw reads that may pass around the same base multiple
times. A raw read could therefore have a structure that is composed of left adapter →DNA insert →
right adapter →reverse complement of DNA insert →left adapter →DNA insert →and so on. This
raw read is typically processed downstream to remove adapters and create subreads composed of the
DNA sequence of interest to the investigator. See the Technical Validation section for details on filtering
parameters and free software used to analyze and quality check the data.
All datasets were filtered and mapped using SMRT Analysis v2.0.1, v2.1.0, or v2.2. There are no
changes to the filtering or mapping parameters in these versions, and detailed parameters used are
discussed in the Technical Validation section. SMRT Analysis is a software suite that is free and can be
downloaded from the Pacific Biosciences Developer’s Community Network Website (DevNet)
37
. SMRT
Analysis includes the SMRT Portal graphical user interface, as well as SMRT View genome browser.
Extensive documentation and technical support can also be accessed via the DevNet website.
The post-filter statistics of each dataset are listed in Table 3. While read lengths reflect the true
sequencing capacity of the instrument, only subreads are summarized in Table 3 because it is relevant and
used for downstream analysis algorithms such as de novo assemblers. Multiple subreads can be contained
within one raw read, and subreads exclude adapters and low quality sequence. N50 is a statistic used to
describe the length distribution of a collection of reads, contigs, or scaffolds, and is defined as the length
where 50% of all bases are contained in sequences longer than that length. The N50 filtered subread
lengths ranged from 7.6–10.5 kb for datasets generated with P4C2 chemistry and ranged from 12.2–14.2
kb for datasets generated with P5C3 chemistry. With the exception of N. crassa OR74A, all datasets were
sequenced to high-coverage (>68X) and sufficient for de novo genome assembly applications. The
N. crassa OR74A dataset was sequenced to 25X coverage and should be sufficient for mapping, consensus
SNP calling, and testing other applications.
Technical Validation
DNA and sample preparation
To assess the quality of genomic DNA received, we used Qbit (Life Technologies) and Nanodrop
(Thermo Scientific) to measure the concentration of genomic DNA. Ideal samples had similar
concentration estimates on both platforms, with A
230/260/230
ratios close to 1:1.8:1, corresponding to what
is expected of pure DNA. All samples presented here passed this screening criterion.
Next we assessed the size of the genomic DNA received. For genomic DNA where the size range was less
than 17 kb, we used the Bioanalyzer 21000 (Agilent) to determine the actual size distribution. For genomic
DNA where the size range was greater than 17 kb, we opted for pulse field gel electrophoresis to better
estimate the larger size distributions. The sizes of the genomic DNA for each sample are listed in Table 1.
Organism Strain Origin Polymerase & Chemistry
Library kits
SRA Accession Size (GB)
E. coli MG1655 Lofstrand Labs P4C2 SRX669475 6.0
E. coli MG1655 Lofstrand Labs P5C3 SRX533603 3.8
S. cerevisiae 9464 J. Li P4C2 SRX533604 38
N. crassa OR74A FGSC P4C2 SRX533605 29
N. crassa T1 D. Catcheside P4C2 SRX533606 143
A thaliana Ler-0 Lehle Seeds P4C2 SRX533608 263
A. thaliana Ler-0 Lehle Seeds P5C3 SRX533607 252
D. melanogaster ISO1 S. Celniker P5C3 SRX499318 187
Table 2. Summary of datasets. Eight datasets from five organisms are described in this paper. Data can
be accessed from SRA using the accession numbers provided.
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SCIENTIFIC DATA |1:140045 |DOI: 10.1038/sdata.2014.45 5
To ensure that the library insert sizes were in the optimal size range, we sheared genomic DNA using
gTubes if the apparent size was greater than 40 kb. Alternatively, if the size was less than 40 kb, then the
DNA was not sheared and carried straight through to library preparation. Extremely small fragments
(o100 bp) and adapter dimers are eliminated by Ampure Beads. Adapter dimers (0–10 bp) and small
inserts (11–100 bp) represented less than 0.01% of all the reads sequenced in all datasets. We additionally
use the Blue Pippin (Sage Science) to ensure that the libraries had a physical size of 10 kb or greater. The
size cutoffs used for each sample are listed in Table 1.
Analysis and quality filtering
To assess the quality of the libraries sequenced, we examined the percent of bases filtered by a standard
QC procedure. Filtering conditions for high-quality SMRT sequence data are read score >0.8, read length
>500 nt, subread length >500 nt. In addition, the ends of reads are trimmed if they are outside of high-
quality (HQ) regions, and adapter sequences between subreads are removed. All samples retained
71–97% of the bases after filtering. High-quality regions are defined by the base caller in primary analysis
(on the PacBio RS II instrument) and indicate the contiguous region of the trace that contains high
quality sequence data
38
. All datasets were filtered with the parameters above using SMRT Analysis v2.0.1,
v2.1.0, or v2.2.0. There are no changes to the filtering protocol in these versions.
To ensure that the sequences matched the model organism of interest, we examined the percent
of post-filter bases that were mapped to the closest reference genome available. All datasets were
mapped using blasr
11
from SMRT Analysis v2.2.0. Plots showing the distributions of mapped
subread concordances are provided in Figure 1, and are a rough measure of how well the reads agree
with the reference genome. Note that these numbers are an underestimate of the true accuracies
of the reads because the DNA was not always from the same strain as the reference genome, and
new stock may have evolved such that certain bases or structural repeats are different from the
reference strain that was first sequenced decades ago. The depth of coverage for each chromosome is also
plotted in Figure 1.
We used reference genomes that were from the closest strain that was available in the public domain.
For E. coli, we used the sequence from NC_000913 at NCBI GenBank, which was first sequenced by
Blattner et al.
39
(Data Citation 2). For S. cerevisae, we downloaded release R64–1–1 of the S288c reference
from the Saccharomyces Genome Database Project (http://downloads.yeastgenome.org/sequence/
S288C_reference/genome_releases/) (Data Citation 3). The changes and updates to the reference genome
are reviewed by Engel et al.
40
For N. crassa, we downloaded the genome sequence from the N. crassa
database hosted by the Broad institute, which uses the same sequences contained in the AABX00000000.3
project at NCBI GenBank (Data Citation 4). This data was from the OR74A strain and first sequenced by
Galagan et al.
41
For A. thaliana, we used the reference sequences from The Arabidopsis Information
Resource
42
(Data Citation 5) (TAIR) version 10 (ftp://ftp.arabidopsis.org/home/tair/Sequences/whole_-
chromosomes/), which was originally sequenced and analyzed by The Arabidopsis Genome Initiative
43
.
For D. melanogaster, we used sequences from RELEASE 5 of the Drosophila reference genome
downloaded from the Berkeley Drosophila Genome Project website (http://www.fruitfly.org/sequence/
release5genomic.shtml). This data was from the ISO1 strain and has been updated since the referenced
RELEASE 3 version by Celniker et al.
29
(Data Citation 6).
All samples had a mapping rate of 81–95%, with the exception of the Neurospora T1 sample that had a
mapping rate of 62%. This sample may have some damaged DNA as it had been stored in a freezer for
over 20 years. Nonetheless, preliminary unpublished results show that the sequence from the Neurospora
T1 sample can be successfully assembled into a genome that is more contiguous than the existing
Dataset Name Number of filtered
subreads
N50 filtered subread
length (nt)
Maximum filtered subread
length (nt)
Total filtered
subread (nt)
Estimated genome
size (Mb)
Fold coverage
E. coli MG1655 P4C2 61,019 7,586 22,609 331,516,965 5 66X
E. coli MG1655 P5C3 43,063 12,041 28,647 373,874,428 5 75X
S. cerevisiae 9464 P4C2 269,145 8,821 30,164 1,597,871,118 12 133X
N. crassa OR74A P4C2 175,926 7,617 30,845 981,884,113 40 25X
N. crassa T1 P4C2 210,480 10,462 36,227 11,497,185,440 40 287X
A. thaliana Ler-0 P4C2 1,338,320 8,769 41,753 8,129,670,483 120 68X
A. thaliana Ler-0 P5C3 2,067,212 12,188 47,445 17,714,447,516 120 148X
D. melanogaster ISO1 P5C3 1,561,929 14,214 44,766 15,194,174,294 160 95X
Table 3. Summary statistics of filtered data. Results shown for each dataset are based on output of
SMRT Portal analysis using the default filtering parameters (see text for details). Fold coverage is calculated
relative to the estimated genome size.
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SCIENTIFIC DATA |1:140045 |DOI: 10.1038/sdata.2014.45 6
reference genome for Neurospora
44
(http://figshare.com/articles/ENCODE_like_study_using_PacBio_
sequencing/928630).
Usage Notes
The eight datasets are available for download in two locations: (1) Amazon S3 repositories, which contain
primary analysis data in the original formats provided by the PacBio RS II instrument (*.metadata.xml, *.
bas.h5, & *.bax.h5 files), and (2) Sequence Read archive entries, which contain unfiltered subread base
calls in sra format and can be converted to unfiltered subread fasta and fastq files using the SRA toolkit.
Links to Amazon S3 repositories as well as SRA accession numbers are provided in Supplementary
Table 1. While fastq files can be used to assess basic read characteristics and analyzed by most third-party
tools, original formats are still needed for more sophisticated analyses using SMRT Portal or PacBio-
specific algorithms. Download the primary data from Amazon S3 if applications such as Quiver
consensus base calling or base modification analyses are desired. These analyses require additional
information encoded within.bax.h5 files such as quality values, pulse width, and inter-pulse duration.
While bax.h5 files can also be converted to fasta or fastq formats, download data from the SRA if sra,
fasta, or fastq formatted files are desired.
The sequence IDs provided in the original formats (bax.h5 files) are different from those provided
by the SRA in sra or fastq formats. The sequence IDs in the original formats contain information
E. coli MG1655 P4C2
coverage
count (1000)
0
1
2
50
concordance
0.70 0.80 0.90 1.00
0
1
2
3
4
# subreads (1000)
S. cerevisiae 9464 P4C2
coverage
0 100 200
chrI
chrII
chrIII
chrIV
chrIX
chrV
chrVI
chrVII
chrVIII
chrX
chrXI
chrXII
chrXIII
chrXIV
chrXV
chrXVI
mitochondrion
D. melanogaster ISO1 P5C3 chromosome
chr2L
chr2LHet
chr2R
chr2RHet
chr3L
chr3LHet
chr3R
chr3RHet
chr4
chrM
chrU
chrUextra
chrX
chrXHet
chrYHet
0 100 200
coverage
count (1000)
0
1
2
count (1000)
0
25
50
75
100
chromosome
concordance
0.70 0.80 0.90 1.00
0
4
8
12
# subreads (1000)
concordance
0.70 0.80 0.90 1.00
0
40
80
120
# subreads (1000)
100
Figure 1. Mapped Subread Concordance and Coverage. The distribution of mapped subread concordances
and mapped subread coverages are plotted for E. coli MG1655 P4C2 (a), S. cerevisiae 9464 P4C2 (b), and
D. melanogaster ISO1 P5C3 (c). The coverage distribution is similar among all chromosomes in S. cerevisiae,
whereas the coverage distribution is half in chrX (50X) compared to the autosomes (100X) in
D. melanogaster. ChrU and chrUextra are assembled contigs that could not be placed to physical
chromosomes, and have very low coverages in general.
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SCIENTIFIC DATA |1:140045 |DOI: 10.1038/sdata.2014.45 7
about the sequencing run itself. The date, time and instrument id is tracked by a ‘m’prefix; the
SMRT Cell barcode, 8 pack number, and other information is tracked by a ‘c’prefix; and the ‘s’and ‘p’
prefixes are now deprecated. For example, a subread with the ID ‘m130227_130322_
42141_c100505662540000001823074808081362_s1_p0/234/3_13049’indicates
that the sequencing run was started in Feburary 27th 2013 (m130227) at 1:03:22PM (130322) on
instrument number 42141, using SMRT Cell ID c100505662540000001823074808081362. This subread
also originates from zero mode wave guide (ZMW) number 234 on the SMRT cell, and corresponds to
bases 3–13049 of the raw read in the ZMW. The same read will be re-named by SRA to an arbitrary index
prefixed by the SRR accession number, and the header line in the fasta file will, for example, appear as
‘>SRR1284620.6 length =13046’in the files downloaded from the SRA.
The datasets described in this paper were first released on DevNet
37
, the PacBio Software Developer
Community Network website, with brief descriptions on the PacBio blog. DevNet typically hosts open-
source software, while SampleNet
27
, the PacBio Sample Preparation Community Network website,
typically hosts protocols for DNA extraction and library preparation. These websites provide valuable
data and documentation about the technology, but are not considered a part of the traditional academic
record. This Data Descriptor in Scientific Data provides an opportunity to describe the methodology and
characteristics of the eight datasets in more detail and creates a citable entity for the scientific community.
DNA sequencing instruments and chemistries change rapidly, and PacBio SMRT sequencing is no
exception. The datasets presented here are from P4C2 and P5C3 polymerase-chemistry combinations,
spanning release dates from late-2013 to early-2014. These datasets represent some of the longest read
lengths to date for these chemistries, and can be used to benchmark and develop new algorithms and the
state of the art as the technology evolves.
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Acknowledgements
The contributions of AMP were funded under Agreement No. HSHQDC-07-C-00020 awarded by
the Department of Homeland Security Science and Technology Directorate (DHS/S&T) for the
management and operation of the National Biodefense Analysis and Countermeasures Center (NBACC),
a Federally Funded Research and Development Center. The views and conclusions contained in this
document are those of the authors and should not be interpreted as necessarily representing the official
policies, either expressed or implied, of the U.S. Department of Homeland Security. In no event shall the
DHS, NBACC, or Battelle National Biodefense Institute (BNBI) have any responsibility or liability for any
use, misuse, inability to use, or reliance upon the information contained herein. The Department of
Homeland Security does not endorse any products or commercial services mentioned in this publication.
CMB was supported by Human Frontier Science Program Young Investigator grant RGY0093/2012.
We thank J. Korlach and E. Hauw for assistance in manuscript preparation, R. Stainer for Neurospora
T1 sample preparation, and J. Trow at NCBI for assistance with data submission.
Author Contributions
K.E.K. prepared libraries, sequenced, and analyzed data for the N. crassa OR74A, N. crassa T1, and
D. melanogaster datasets. P.P. grew plants from seed, prepared libraries, and sequenced DNA for the
A. thaliana P4C2 and A. thaliana P5C3 datasets. He also sequenced DNA for the S. cerevisae 9464
dataset. P.B. prepared libraries and sequenced the E. coli datasets. P.J.Y. provided DNA for the N. crassa
T1 dataset. C.Y. extracted DNA for the D. melanogaster dataset. W.F. collected male flies for the
D. melanogaster dataset. C.-S.C. analyzed data. N.A.R. extracted DNA, prepared libraries, and
coordinated the S. cerevisae 9464 dataset. D.R.R. grew plants from seed, extracted DNA and coordinated
the A. thaliana P4C2 and P5C3 datas000ets. J.L. extracted DNA and prepared libraries for the S. cerevisae
9464 dataset. D.C. provided DNA for the N. crassa T1 dataset. S.E.C. extracted DNA and coordinated the
D. melanogaster dataset. A.M.P. analyzed data, coordinated the project, and prepared the manuscript.
C.M.B. analyzed data, coordinated the project, and prepared the manuscript. J.M.L. deposited data to the
SRA, analyzed data, coordinated the project, and prepared the manuscript.
Additional information
Supplementary information accompanies this paper at http://www.nature.com/sdata
Competing financial interests: The authors declare competing financial interests. K.E.K., P.P., P.B.,
C.-S.C., N.A.R., D.R.R., and J.M.L. are employees of Pacific Biosciences of California, Inc., a company
commercializing DNA sequencing technologies.
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SCIENTIFIC DATA |1:140045 |DOI: 10.1038/sdata.2014.45 9
How to cite this article: Kim, K. E. et al. Long-read, whole-genome shotgun sequence data for five model
organisms. Sci. Data 1:140045 doi: 10.1038/sdata.2014.45 (2014).
This work is licensed under a Creative Commons Attribution 4.0 International License. The
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Creative Commons license, users will need to obtain permission from the license holder to reproduce the
material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0
Metadata associated with this Data Descriptor is available at http://www.nature.com/sdata/ and is released
under the CC0 waiver to maximize reuse.
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