Abstract Sample prep and sequencing - lanl.gov
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Los Alamos National Laboratory PacBio-based Assemblies of Small Eukaryotes Yuliya Kunde*, Karen Davenport, Cheryl Gleasner, Kim McMurry, Olga Chertkov, Shawn Starkenburg Los Alamos NaDonal Laboratory, Los Alamos, NM PhotosyntheDc microalgae are a promising source of feedstock material for biofuels. The mechanism(s) of lipid producDon are not fully understood, but the widely accepted hypothesis is that under stress condiDons, microalgae convert excess energy from light into storage compounds like starch and lipids. Currently, high quality genome assemblies from microalgae producDon strains are not available (thousands of conDgs). Access to nearly finished genomes for these organisms will significantly improve our understanding of key metabolic pathways, and inform raDonal geneDc engineering approaches. For this purpose, genomic DNA from two top ranking candidates, Chlorella sp. (strains 1228 and 1230) and Scenedesmus obliquus, was converted into 20kb libraries for sequencing and assembly with PacBio and HGAP, respecDvely. The PacBiobased assemblies were further improved with short reads from Illumina or OpGen opDcal maps. Herein, we will present the comparisons of these assembly methods as well as costbenefit analysis of generaDng hybrid assemblies with PacBio and OpGen. Abstract Sample prep and sequencing . Data analysis and genome assembly The ability of microalgae to store lipids under stress makes them an aXracDve source for the producDon of biofuels. Unfortunately, liXle is known about metabolic pathways that lead to the lipid storage. Shedding light on these biochemical transformaDons will significantly improve our understanding of the lifecycle of these organisms and will allow for efficient manipulaDons in growing and harvesDng algal biomass. DNA sequencing is a powerful tool in reaching this goal. While typical NGS technologies can provide plenty of sequencing data, their short reads and amplificaDon biases will produce fragmented genome assemblies whenever a complex repeat or poorly amplified (high or low GC) regions are encountered. Resolving fragmented assemblies will require more costly laboratory and computaDonal resources. Using PacBio long reads will reduce the Dme and simplify genome finishing process. We used various PacBiobased approaches to analyze data and perform genome assembly: Long PacBio reads data (HGAP assembly) was supplemented with the OpGen opDcal maps for C. sorokiniana 1228 and manual work brought the genome to the final assembly. Long PacBio reads data (HGAP assembly) was used alone or in combinaDon with short reads data assemblies for C. sorokiniana 1230 and manual work interspersed through the processes and a_er merging the assemblies brought the genome to the final assembly. Long PacBio reads data only (HGAP assembly) was used to analyze and assemble the S. obliquus genome. 9,652 7,477 2,753 1,147 64 82 20 0 2,000 4,000 6,000 8,000 10,000 12,000 A B C D E F G Number of con>gs Con>g Number 11,009 15,504 44,845 98,320 2,415,094 3,828,126 4,091,730 0 500,000 1,000,000 1,500,000 2,000,000 2,500,000 3,000,000 3,500,000 4,000,000 4,500,000 A B C D E F G Number of base pairs Con>g N50 84,533 116,504 236,940 435,074 4,567,720 5,130,349 5,120,617 0 1,000,000 2,000,000 3,000,000 4,000,000 5,000,000 6,000,000 A B C D E F G Number of base pairs Maximum Con>g 54,829,407 56,036,303 56,020,885 56,521,810 61,391,260 59,715,620 58,534,920 50,000,000 52,000,000 54,000,000 56,000,000 58,000,000 60,000,000 62,000,000 A B C D E F G Number of base pairs Assembly Size Figure 4. Algal Genome Assembly and improvement Processes for C. sorokiniana 1230: Illumina data, single kmerbased assembly; B. Assembly A improved with manual computaDonal work; C. Assembly from a collaborator (unknown shortread data type, assembly type and improvement process); D. Assemblies B and C merged; E. Assembly D improved with manual computaDonal work; F. PacBio data, overlap assembly; G. Assemblies E and F merged and improved with manual computaDonal work. Illuminaonly assemblies are shown in red. PacBioonly assemblies are shown in blue. The collaborator’s assembly is shown in gray. Merged assemblies are shown striped. The final assembly was a near finished genome (20 conDgs with two completed circular organellar chromosomes with the algal chromosome completed telomere to telomere with only a few gaps). Three algal candidates for the producDon of biofuels were sequenced at LANL using Pacific Biosciences NGS plaform RSII. PacBio is the current standard for long read data and is certainly advantageous for genomic analysis of eukaryotes. As shown in Figure 3, the OpGen map provided excellent scaffolding for the PacBio conDgs, but the OpGen/ PacBio combinaDon for C. sorokiniana 1228 was not as complete (64 conDgs) as the short read/long read merged assemblies of C. sorokiniana 1230 (20 conDgs). While the OpGen technology is expensive, the manual work on the short reads assemblies is also expensive and is likely of similar cost. The HGAP assembly of PacBioonly data for the S. obliquus genome was problemaDc, since the genome size produced was double what was expected. We are experimenDng with FALCON (an assembler designed for higher ploidy/larger genomes) for the assembly of the S. obliquus genome due to the heterozygosity in this genome. IniDal results with FALCON provide a reduced genome size (closer to the 100 Mb expected) and with a reducDon in conDg number by 2530%. These assemblies indicate 1520% of the genome is heterozygous. All of this PacBio data were produced with the P5/C3 chemistry; we believe that if generated with the current P6/C4 chemistry (or the soontobe released chemistry with longer movie Dmes) the iniDal PacBioonly assemblies would be beXer and the costs would be significantly reduced. Algae species Es>mated genome size (Mbp) # of template preps # of SMRT cells sequenced # of polished con>gs in HGAP Genome coverage by PacBio # of Mbp of data Template prep and sequencing cost C. sorokiniana 1228 61 3 41 322 38x 6,083 $14,860.97 C. sorokiniana 1230 58 1 16 85 85x 5,866 $7,341.15 S. obliquus 210 2 46 3,081 57x 18,238 $15,870.94 Algae species Technologies Es>mated genome size (Mbp) Final Con>gs Final N50 Final Largest Con>g Final Assembly Size C. sorokiniana 1228 PacBio, OpGen 60 64 2,415,094 4,567,720 61,391,260 C. sorokiniana 1230 PacBio, Illumina 55 20 4,091,730 5,120,617 58,534,920 S. obliquus PacBio only 100* 2812 152,111 2,334,183 210,263,644 Figure 1. Sequencing results and costs for three algal genomes. All three have 20 Kbp prep libraries. Figure 2. Final assembly results for three algal genomes. *While it’s thought that S. obliquus genome size is 100 Mbp, this assembly has a size of 200 Mbp which could be a result of heterozygosity. Figure 3. OpDcal maps for C. sorokiniana 1228 with HGAP conDgs (>40kb) scaffolded into 12 algal chromosomes (telomere to telomere). Some manual work on the 12 chromosomal scaffolds produced a final assembly of 64 conDgs and an assembly size of 61 Mbp. This included two smaller circular organellar chromosomes which were completed in the HGAP assembly. Introduction Conclusions Algal cells of C. sorokiniana 1228 were grown in culture, harvested and imbedded into the agarose plugs for further extracDon of the HMW DNA. The DNA was extracted through enzymaDc removal( destrucDon) of the cell wall and subsequent lysis of the cells. Some of the algal plugs were sent to OpGen for opDcal mapping, the others were used for the extracDon of HMW genomic DNA and preparaDon of 20kb SMRT bell library according to the standard PacBio protocol. Genomic DNA for S. obliquus and C. sorokiniana 1230 was received from an outside collaborator and processed for 20kb SMRT bell library prep. The long insert libraries were sizeselected using Blue Pippin instrument. The sequencing primer was annealed to the selected SMRT bells. DNA polymerase was bound to the SMRT bell template and the samples were loaded onto the SMRT cells using MagBeads. The samples were sequenced with C3P5 chemistry and 3 hour movies. The short reads data was generated both at LANL and by an outside collaborator
Transcript of Abstract Sample prep and sequencing - lanl.gov
Los Alamos National Laboratory
PacBio-based Assemblies of Small Eukaryotes Yuliya Kunde*, Karen Davenport, Cheryl Gleasner, Kim McMurry, Olga Chertkov, Shawn Starkenburg
Los Alamos NaDonal Laboratory, Los Alamos, NM
PhotosyntheDc microalgae are a promising source of feedstock material for biofuels. The mechanism(s) of lipid producDon are not fully understood, but the widely accepted hypothesis is that under stress condiDons, microalgae convert excess energy from light into storage compounds like starch and lipids. Currently, high quality genome assemblies from microalgae producDon strains are not available (thousands of conDgs). Access to nearly finished genomes for these organisms will significantly improve our understanding of key metabolic pathways, and inform raDonal geneDc engineering approaches. For this purpose, genomic DNA from two top ranking candidates, Chlorella sp. (strains 1228 and 1230) and Scenedesmus obliquus, was converted into 20kb libraries for sequencing and assembly with PacBio and HGAP, respecDvely. The PacBio-‐based assemblies were further improved with short reads from Illumina or OpGen opDcal maps. Herein, we will present the comparisons of these assembly methods as well as cost-‐benefit analysis of generaDng hybrid assemblies with PacBio and OpGen.
Abstract Sample prep and sequencing
.
Data analysis and genome assembly
The ability of microalgae to store lipids under stress makes them an aXracDve source for the producDon of biofuels. Unfortunately, liXle is known about metabolic pathways that lead to the lipid storage. Shedding light on these biochemical transformaDons will significantly improve our understanding of the lifecycle of these organisms and will allow for efficient manipulaDons in growing and harvesDng algal biomass. DNA sequencing is a powerful tool in reaching this goal. While typical NGS technologies can provide plenty of sequencing data, their short reads and amplificaDon biases will produce fragmented genome assemblies whenever a complex repeat or poorly amplified (high or low GC) regions are encountered. Resolving fragmented assemblies will require more costly laboratory and computaDonal resources. Using PacBio long reads will reduce the Dme and simplify genome finishing process.
We used various PacBio-‐based approaches to analyze data and perform genome assembly: ² Long PacBio reads data (HGAP assembly) was supplemented with the OpGen
opDcal maps for C. sorokiniana 1228 and manual work brought the genome to the final assembly.
² Long PacBio reads data (HGAP assembly) was used alone or in combinaDon with short reads data assemblies for C. sorokiniana 1230 and manual work interspersed through the processes and a_er merging the assemblies brought the genome to the final assembly.
² Long PacBio reads data only (HGAP assembly) was used to analyze and assemble the S. obliquus genome.
9,652
7,477
2,753
1,147
64 82 20 0
2,000
4,000
6,000
8,000
10,000
12,000
A B C D E F G
Num
ber o
f con
>gs
Con>g Number
11,009 15,504 44,845 98,320
2,415,094
3,828,126 4,091,730
0
500,000
1,000,000
1,500,000
2,000,000
2,500,000
3,000,000
3,500,000
4,000,000
4,500,000
A B C D E F G
Num
ber o
f base pa
irs
Con>g N50
84,533 116,504 236,940 435,074
4,567,720
5,130,349 5,120,617
0
1,000,000
2,000,000
3,000,000
4,000,000
5,000,000
6,000,000
A B C D E F G
Num
ber o
f base pa
irs
Maximum Con>g
54,829,407
56,036,303 56,020,885
56,521,810
61,391,260
59,715,620
58,534,920
50,000,000
52,000,000
54,000,000
56,000,000
58,000,000
60,000,000
62,000,000
A B C D E F G
Num
ber o
f base pa
irs
Assembly Size
Figure 4. Algal Genome Assembly and improvement Processes for C. sorokiniana 1230: Illumina data, single kmer-‐based assembly; B. Assembly A improved with manual computaDonal work; C. Assembly from a collaborator (unknown short-‐read data type, assembly type and improvement process); D. Assemblies B and C merged; E. Assembly D improved with manual computaDonal work; F. PacBio data, overlap assembly; G. Assemblies E and F merged and improved with manual computaDonal work. Illumina-‐only assemblies are shown in red. PacBio-‐only assemblies are shown in blue. The collaborator’s assembly is shown in gray. Merged assemblies are shown striped. The final assembly was a near finished genome (20 conDgs with two completed circular organellar chromosomes with the algal chromosome completed telomere to telomere with only a few gaps).
Three algal candidates for the producDon of biofuels were sequenced at LANL using Pacific Biosciences NGS plaform RSII. PacBio is the current standard for long read data and is certainly advantageous for genomic analysis of eukaryotes. As shown in Figure 3, the OpGen map provided excellent scaffolding for the PacBio conDgs, but the OpGen/PacBio combinaDon for C. sorokiniana 1228 was not as complete (64 conDgs) as the short read/long read merged assemblies of C. sorokiniana 1230 (20 conDgs). While the OpGen technology is expensive, the manual work on the short reads assemblies is also expensive and is likely of similar cost. The HGAP assembly of PacBio-‐only data for the S. obliquus genome was problemaDc, since the genome size produced was double what was expected. We are experimenDng with FALCON (an assembler designed for higher ploidy/larger genomes) for the assembly of the S. obliquus genome due to the heterozygosity in this genome. IniDal results with FALCON provide a reduced genome size (closer to the 100 Mb expected) and with a reducDon in conDg number by 25-‐30%. These assemblies indicate 15-‐20% of the genome is heterozygous. All of this PacBio data were produced with the P5/C3 chemistry; we believe that if generated with the current P6/C4 chemistry (or the soon-‐to-‐be released chemistry with longer movie Dmes) the iniDal PacBio-‐only assemblies would be beXer and the costs would be significantly reduced.
Algae species Es>mated genome size
(Mbp)
# of template preps
# of SMRT cells
sequenced
# of polished con>gs in HGAP
Genome coverage by PacBio
# of Mbp of data
Template prep and sequencing cost
C. sorokiniana 1228 61 3 41 322 38x 6,083 $14,860.97
C. sorokiniana 1230 58 1 16 85 85x 5,866 $7,341.15
S. obliquus 210 2 46 3,081 57x 18,238 $15,870.94
Algae species Technologies Es>mated genome size (Mbp)
Final Con>gs Final N50 Final Largest
Con>g Final Assembly
Size
C. sorokiniana 1228 PacBio, OpGen 60 64 2,415,094 4,567,720 61,391,260
C. sorokiniana 1230 PacBio, Illumina 55 20 4,091,730 5,120,617 58,534,920
S. obliquus PacBio only 100* 2812 152,111 2,334,183 210,263,644
Figure 1. Sequencing results and costs for three algal genomes. All three have 20 Kbp prep libraries.
Figure 2. Final assembly results for three algal genomes. *While it’s thought that S. obliquus genome size is 100 Mbp, this assembly has a size of 200 Mbp which could be a result of heterozygosity.
Figure 3. OpDcal maps for C. sorokiniana 1228 with HGAP conDgs (>40kb) scaffolded into 12 algal chromosomes (telomere to telomere). Some manual work on the 12 chromosomal scaffolds produced a final assembly of 64 conDgs and an assembly size of 61 Mbp. This included two smaller circular organellar chromosomes which were completed in the HGAP assembly.
Introduction
Conclusions
Algal cells of C. sorokiniana 1228 were grown in culture, harvested and imbedded into the agarose plugs for further extracDon of the HMW DNA. The DNA was extracted through enzymaDc removal( destrucDon) of the cell wall and subsequent lysis of the cells. Some of the algal plugs were sent to OpGen for opDcal mapping, the others were used for the extracDon of HMW genomic DNA and preparaDon of 20kb SMRT bell library according to the standard PacBio protocol. Genomic DNA for S. obliquus and C. sorokiniana 1230 was received from an outside collaborator and processed for 20kb SMRT bell library prep. The long insert libraries were size-‐selected using Blue Pippin instrument. The sequencing primer was annealed to the selected SMRT bells. DNA polymerase was bound to the SMRT bell template and the samples were loaded onto the SMRT cells using MagBeads. The samples were sequenced with C3-‐P5 chemistry and 3 hour movies. The short reads data was generated both at LANL and by an outside collaborator
