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The US DOE Joint Genome Institute (JGI) is a high-throughput genomics facility involved in sequencing a variety of organisms. A major effort at JGI is the sequencing of genomes and microbial community samples of relevance to the DOE missions of carbon sequestration, bioremediation and energy production. cDNA/EST sequencing is an integral part of genomic sequencing because it provides crucial information for gene models and genome annotation. The 454 sequencing platform is an integrated system of emulsion-based PCR amplification of hundreds of thousands of DNA fragments linked to high throughput parallel pyrosequencing in picoliter-sized wells. Several strategies have been designed and carried out at JGI to use the 454 platform for cDNA/EST sequencing. cDNA libraries constructed by conventional methods were subjected to direct 454 sequencing. In addition, special primers and adaptors were also designed for library construction so the directional sequencing feature of the 454 technology can be used to sequence a particular end of the cDNA/EST fragments. Adaptor sequences used by 454 library construction can be incorporated into polyT primer, cap primer and/or random primer for cDNA/EST library construction. The 454 sequencing platform can deliver 200 to 400 thousand cDNA/EST reads from a single run and does not require cloning step, potentially improving the coverage obtained through traditional Sanger sequencing. The large numbers of short reads generated by the 454 platform can be aligned to genome assemblies to extend and confirm gene models. Results from different strategies of library construction combined with 454 sequencing will be presented. The coverage of the library and the novelty rate are compared with traditional Sanger sequencing. The possible assembly problems caused by short reads with slightly higher error rate from 454 will also be addressed.

The Department of Energy Joint Genome Institute (www.jgi.doe.gov) in Walnut Creek, CA is a high throughput DNA sequencing facility with a current throughput of approximately 3 billion Sanger base pairs per month. A major effort at JGI is the sequencing of microbial genomes of relevance to the DOE missions of carbon sequestration, bioremediation and energy production. The JGI Microbial Program and Community Sequencing Program together are responsible for the generation of sequencing data for over 400 microbial genomes. At the traditional Sanger sequencing side, JGI is running about 70 ABI sequencers on a 24/7 schedule and about 40 GE MegaBACE 4500 sequencers on a 24/5 schedule. JGI currently runs 2 Roche's GS20 instruments to supplement our traditional Sanger sequencing. Our current whole genome shotgun sequencing strategy is to sequence 3kb and/or 8kb shotgun libraries to a combined 4-8x draft coverage and to sequence fosmid ends to 1x sequence coverage with Sanger sequencing and to supplement that with 12-25x coverage with 454 sequencing platform depending on the sizes of the genomes. For new microbial genomes we initiated, 454 sequencing was carried out at the same time the shotgun cloning for Sanger sequencing started. 454 sequencing data was used to profile the genomes for G/C content, genome sizes and other features of the genomes. For existing microbial genome projects for which the Sanger sequencing data has already been generated, we have been adding 454 sequencing coverage at the finishing stage. 454 sequencing data was assembled by default Newbler assembler software package from 454 Life Sciences. The Newbler contigs were then fragmented and the quality and coverage information of the contigs was captured by in-house developed software tool packages. The fragmentation strategy we currently use is to cut the Newbler contigs into 750 bp fragments with 100 bp overlap. The overlapping fragments from Newbler contigs were finally assembled with Sanger sequencing

SprintPrep DNA isolation is a process that allows large fragments of DNA and vectors to be isolated from the host E. Coli cell. Agencourt has developed SprintPrep reagents and semi-automated methods for performing the necessary protocol. Last year, JGI implemented a 96 well SprintPrep method. This year, JGI has made the 384 SprintPrep method virtually user-independent. Moving from the 96 well fosmid isolation method to the 384 well format has led to cost savings due to reagent reductions and a doubling in sequencing throughput. The increase in throughput will lead to an increase in sequencing depth and data confidence.

High quality fosmid end-sequencing plays an important role in whole genome shotgun assembly. Accurate paired end information at the size of about 40 kb is crucial in building large genome scaffolds. We have developed an automated high-throughput fosmid DNA isolation and sequencing protocol using a magnetic bead prep (Agencourt) and terminator cycling sequencing. This method uses 384-well format plates from cell growth, DNA isolation to sequencer loading, significantly increases the throughput comparing to themethod using 96-well format plates. Using Beckman s Biomek FX with dual pods but without stacker carousel, our throughput is 8 384-well plates in less than 2 hours per instrument. After the fosmid DNA is eluted, cycling sequencing was performed using reduced reagents and according to our standard production protocol. We are able to achieve a pass rate (Q20 > 50) of over 95% and average read length (Q20) over 650 bp. Next steps will be to utilize stacker carousels to double our throughput to 16 plates in same amount of time and to further reduce sequencing reagents while maintaining high quality.

At JGI, major efforts have been spent on sequencing through gaps in the assemblies generated by whole genome shotgun sequencing. Traditional shotgun sequencing is known to have difficulty in both cloning of A/T rich regions and sequencing of G/C rich regions. To help alleviate this problem we have applied the 454 sequencing platform as another tool for gap closure. Although 454 sequencing has been shown to have difficulty with homopolymer, it does not have the same biases as traditional sequencing. Therefore these two approaches together can be complementary. Different strategies have been tested for applying 454 sequencing in gap closure stage of WGS sequencing. Direct shotgun sequencing using 454 platform has been combined with traditional Sanger sequencing at the final assembly stage. We also developed a protocol in which gap-spanning clones are pooled, sequenced and assembled with 454 platform and the resulting contigs are added into their respective Sanger assemblies. The quality of the final assemblies from these different strategies was examined and compared. The regions only covered by 454 sequencing were studied and the sequencing features of these regions were analyzed. The base quality and assembly correctness of these regions were also assessed. Detailed results will be presented.

The Department of Energy Joint Genome Institute (www.jgi.doe.gov) in Walnut Creek, CA is a high throughput DNA sequencing facility with a current throughput of approximately 3 billion Sanger base pairs per month. A major effort at JGI is the sequencing of microbial genomes of relevance to the DOE missions of carbon sequestration, bioremediation and energy production. The JGI Microbial Program and Community Sequencing Program together are responsible for the generation of sequencing data for over 400 microbial genomes. At the traditional Sanger sequencing side, JGI is running about 70 ABI sequencers on a 24/7 schedule and about 40 GE MegaBACE 4500 sequencers on a 24/5 schedule. JGI currently runs 2 Roche's GS20 instruments to supplement our traditional Sanger sequencing. Our current whole genome shotgun sequencing strategy is to sequence 3kb and/or 8kb shotgun libraries to a combined 4-8x draft coverage and to sequence fosmid ends to 1x sequence coverage with Sanger sequencing and to supplement that with 12-25x coverage with 454 sequencing platform depending on the sizes of the genomes. For new microbial genomes we initiated, 454 sequencing was carried out at the same time the shotgun cloning for Sanger sequencing started. 454 sequencing data was used to profile the genomes for G/C content, genome sizes and other features of the genomes. For existing microbial genome projects for which the Sanger sequencing data has already been generated, we have been adding 454 sequencing coverage at the finishing stage. 454 sequencing data was assembled by default Newbler assembler software package from 454 Life Sciences. The Newbler contigs were then fragmented and the quality and coverage information of the contigs was captured by in-house developed software tool packages. The fragmentation strategy we currently use is to cut the Newbler contigs into 750 bp fragments with 100 bp overlap. The overlapping fragments from Newbler contigs were finally assembled with Sanger sequencing

The US DOE Joint Genome Institute (JGI) is a high-throughput genomics facility involved in sequencing a variety of organisms. A major effort at JGI is the sequencing of genomes and microbial community samples of relevance to the DOE missions of carbon sequestration, bioremediation and energy production. cDNA/EST sequencing is an integral part of genomic sequencing because it provides crucial information for gene models and genome annotation. The 454 sequencing platform is an integrated system of emulsion-based PCR amplification of hundreds of thousands of DNA fragments linked to high throughput parallel pyrosequencing in picoliter-sized wells. Several strategies have been designed and carried out at JGI to use the 454 platform for cDNA/EST sequencing. cDNA libraries constructed by conventional methods were subjected to direct 454 sequencing. In addition, special primers and adaptors were also designed for library construction so the directional sequencing feature of the 454 technology can be used to sequence a particular end of the cDNA/EST fragments. Adaptor sequences used by 454 library construction can be incorporated into polyT primer, cap primer and/or random primer for cDNA/EST library construction. The 454 sequencing platform can deliver 200 to 400 thousand cDNA/EST reads from a single run and does not require cloning step, potentially improving the coverage obtained through traditional Sanger sequencing. The large numbers of short reads generated by the 454 platform can be aligned to genome assemblies to extend and confirm gene models. Results from different strategies of library construction combined with 454 sequencing will be presented. The coverage of the library and the novelty rate are compared with traditional Sanger sequencing. The possible assembly problems caused by short reads with slightly higher error rate from 454 will also be addressed.

SprintPrep DNA isolation is a process that allows large fragments of DNA and vectors to be isolated from the host E. Coli cell. Agencourt has developed SprintPrep reagents and semi-automated methods for performing the necessary protocol. Last year, JGI implemented a 96 well SprintPrep method. This year, JGI has made the 384 SprintPrep method virtually user-independent. Moving from the 96 well fosmid isolation method to the 384 well format has led to cost savings due to reagent reductions and a doubling in sequencing throughput. The increase in throughput will lead to an increase in sequencing depth and data confidence.

At JGI, major efforts have been spent on sequencing through gaps in the assemblies generated by whole genome shotgun sequencing. Traditional shotgun sequencing is known to have difficulty in both cloning of A/T rich regions and sequencing of G/C rich regions. To help alleviate this problem we have applied the 454 sequencing platform as another tool for gap closure. Although 454 sequencing has been shown to have difficulty with homopolymer, it does not have the same biases as traditional sequencing. Therefore these two approaches together can be complementary. Different strategies have been tested for applying 454 sequencing in gap closure stage of WGS sequencing. Direct shotgun sequencing using 454 platform has been combined with traditional Sanger sequencing at the final assembly stage. We also developed a protocol in which gap-spanning clones are pooled, sequenced and assembled with 454 platform and the resulting contigs are added into their respective Sanger assemblies. The quality of the final assemblies from these different strategies was examined and compared. The regions only covered by 454 sequencing were studied and the sequencing features of these regions were analyzed. The base quality and assembly correctness of these regions were also assessed. Detailed results will be presented.

High quality fosmid end-sequencing plays an important role in whole genome shotgun assembly. Accurate paired end information at the size of about 40 kb is crucial in building large genome scaffolds. We have developed an automated high-throughput fosmid DNA isolation and sequencing protocol using a magnetic bead prep (Agencourt) and terminator cycling sequencing. This method uses 384-well format plates from cell growth, DNA isolation to sequencer loading, significantly increases the throughput comparing to themethod using 96-well format plates. Using Beckman s Biomek FX with dual pods but without stacker carousel, our throughput is 8 384-well plates in less than 2 hours per instrument. After the fosmid DNA is eluted, cycling sequencing was performed using reduced reagents and according to our standard production protocol. We are able to achieve a pass rate (Q20 > 50) of over 95% and average read length (Q20) over 650 bp. Next steps will be to utilize stacker carousels to double our throughput to 16 plates in same amount of time and to further reduce sequencing reagents while maintaining high quality.

High quality fosmid end-sequencing plays an important role in whole genome shotgun assembly. Accurate paired end information at the size of about 40 kb is crucial in building large genome scaffolds. We have developed an automated high-throughput fosmid DNA isolation and sequencing protocol using a magnetic bead prep (Agencourt) and terminator cycling sequencing. This method uses 384-well format plates from cell growth, DNA isolation to sequencer loading, significantly increases the throughput comparing to the method using 96-well format plates. Using Beckman s Biomek FX without stacker carousel, our throughput is 6 384-well plates in 2 hours per instrument. After the fosmid DNA is eluted, cycling sequencing was performed using reduced reagents and according to our standard production protocol. We are able to achieve a pass rate (Q20 > 50) of over 95% and average read length (Q20) over 650 bp. Next steps will be to utilize stacker carousels to double our throughput to 12 plates in same amount of time and to further reduce sequencing reagents while maintaining high quality.

The US DOE Joint Genome Institute (JGI) is a high-throughput sequencing center involved in a myriad of sequencing projects. A major effort at JGI is the sequencing of microbial genomes of relevance to the DOE missions of carbon sequestration, bioremediation and energy production. The JGI Microbial Program is responsible for the generation of over 200 microbial genomes and we are interested in utilizing new technologies to increase capacity. The 454 sequencing platform is an integrated system of emulsion-based PCR amplification of hundreds of thousands of DNA fragments linked to high throughput parallel pyrosequencing in picoliter-sized wells. The 454 sequencing platform can deliver 30 to 50 million base pairs (mbp) from a single run, however, our previous study revealed that the quality of the resulting assembly contains high numbers of misassemblies and base errors due to short read length and lack of paired-end information. The traditional Sanger sequencing method is lower in throughput and more costly but it provides high quality sequencing results and more accurate assemblies. The paired-end information from Sanger sequencing is proven to be crucial in scaffolding and gap closure. We combined 454 sequencing results with different amounts of paired Sanger sequencing reads from three different sized shotgun libraries and analyzed the results. Assemblies from all combinations were done by Newbler and Phred/Phrap and viewed and analyzed by Consed and in-house developed software. Numbers of remaining gaps and low quality regions from different combinations were assessed. Distribution of coverage and possible errors were analyzed for both platforms. We will also discuss the optimal ratio of data from 454 and Sanger sequencing to achieve high quality finished microbial genome sequences in a time and cost effective manner.

Implementing the Agencourt SprintPrep384 Protocol at JGIPresenting Author: Steven E. Wilson Contributing Authors: Paul Richardson, Feng Chen, Jamie Jett, Nancy Hammon, Duane Kubischta, Diana Lawrence U.S. DOE Joint Genome Institute 2800 Mitchell Drive, Bldg. 100 Walnut Creek, CA 94598 sewilson@lbl.gov(925) 296-5769 SprintPrep DNA isolation is a process that allows large fragments of DNA and vectors to be isolated from the host E. Coli cell. Agencourt has developed SprintPrep reagents and semi-automated methods for performing the necessary protocol. Last year, JGI implemented a 96 well SprintPrep method. This year, JGI has made the 384 SprintPrep method virtually user-independent. Moving from the 96 well fosmid isolation method to the 384 well format has led to cost savings due to reagent reductions and a doubling in sequencing throughput. The increase in throughput will lead to an increase in sequencing depth and data confidence.

High quality fosmid end-sequencing plays an important role in whole genome shotgun assembly. Accurate paired end information at the size of about 40 kb is crucial in building large genome scaffolds. We have developed an automated high-throughput fosmid DNA isolation and sequencing protocol using a magnetic bead prep (Agencourt) and terminator cycling sequencing. This method uses 384-well format plates from cell growth, DNA isolation to sequencer loading, significantly increases the throughput comparing to the method using 96-well format plates. Using Beckman s Biomek FX without stacker carousel, our throughput is 6 384-well plates in 2 hours per instrument. After the fosmid DNA is eluted, cycling sequencing was performed using reduced reagents and according to our standard production protocol. We are able to achieve a pass rate (Q20 > 50) of over 95% and average read length (Q20) over 650 bp. Next steps will be to utilize stacker carousels to double our throughput to 12 plates in same amount of time and to further reduce sequencing reagents while maintaining high quality.

The US DOE Joint Genome Institute (JGI) is a high-throughput sequencing center involved in a myriad of sequencing projects. A major effort at JGI is the sequencing of microbial genomes of relevance to the DOE missions of carbon sequestration, bioremediation and energy production. The JGI Microbial Program is responsible for the generation of over 200 microbial genomes and we are interested in utilizing new technologies to increase capacity. The 454 sequencing platform is an integrated system of emulsion-based PCR amplification of hundreds of thousands of DNA fragments linked to high throughput parallel pyrosequencing in picoliter-sized wells. The 454 sequencing platform can deliver 30 to 50 million base pairs (mbp) from a single run, however, our previous study revealed that the quality of the resulting assembly contains high numbers of misassemblies and base errors due to short read length and lack of paired-end information. The traditional Sanger sequencing method is lower in throughput and more costly but it provides high quality sequencing results and more accurate assemblies. The paired-end information from Sanger sequencing is proven to be crucial in scaffolding and gap closure. We combined 454 sequencing results with different amounts of paired Sanger sequencing reads from three different sized shotgun libraries and analyzed the results. Assemblies from all combinations were done by Newbler and Phred/Phrap and viewed and analyzed by Consed and in-house developed software. Numbers of remaining gaps and low quality regions from different combinations were assessed. Distribution of coverage and possible errors were analyzed for both platforms. We will also discuss the optimal ratio of data from 454 and Sanger sequencing to achieve high quality finished microbial genome sequences in a time and cost effective manner.

Implementing the Agencourt SprintPrep384 Protocol at JGIPresenting Author: Steven E. Wilson Contributing Authors: Paul Richardson, Feng Chen, Jamie Jett, Nancy Hammon, Duane Kubischta, Diana Lawrence U.S. DOE Joint Genome Institute 2800 Mitchell Drive, Bldg. 100 Walnut Creek, CA 94598 sewilson@lbl.gov(925) 296-5769 SprintPrep DNA isolation is a process that allows large fragments of DNA and vectors to be isolated from the host E. Coli cell. Agencourt has developed SprintPrep reagents and semi-automated methods for performing the necessary protocol. Last year, JGI implemented a 96 well SprintPrep method. This year, JGI has made the 384 SprintPrep method virtually user-independent. Moving from the 96 well fosmid isolation method to the 384 well format has led to cost savings due to reagent reductions and a doubling in sequencing throughput. The increase in throughput will lead to an increase in sequencing depth and data confidence.

We report here the 78,884,754 base pairs of finished human chromosome 16 sequence, representing over 99.9 percent of its euchromatin. Manual annotation revealed 880 protein coding genes confirmed by 1,637 aligned transcripts, 19 tRNA genes, 341 pseudogenes and 3 RNA pseudogenes. These genes include metallothionein, cadherin and iroquois gene families, as well as the disease genes for polycystic kidney disease and acute myelomonocytic leukemia. Several large-scale structural polymorphisms spanning hundreds of kilobasepairs were identified and result in gene content differences across humans. One of the unique features of chromosome 16 is its high level of segmental duplication, ranked among the highest of the human autosomes. While the segmental duplications are enriched in the relatively gene poor pericentromere of the p-arm, some are involved in recent gene duplication and conversion events which are likely to have had an impact on the evolution of primates and human disease susceptibility.

Chromosome 5 is one of the largest human chromosomes yet has one of the lowest gene densities. This is partially explained by numerous gene-poor regions that display a remarkable degree of noncoding and syntenic conservation with non-mammalian vertebrates, suggesting they are functionally constrained. In total, we compiled 177.7 million base pairs of highly accurate finished sequence containing 923 manually curated protein-encoding genes including the protocadherin and interleukin gene families and the first complete versions of each of the large chromosome 5 specific internal duplications. These duplications are very recent evolutionary events and play a likely mechanistic role, since deletions of these regions are the cause of debilitating disorders including spinal muscular atrophy (SMA).

We report here the 78,884,754 base pairs of finished human chromosome 16 sequence, representing over 99.9 percent of its euchromatin. Manual annotation revealed 880 protein coding genes confirmed by 1,637 aligned transcripts, 19 tRNA genes, 341 pseudogenes and 3 RNA pseudogenes. These genes include metallothionein, cadherin and iroquois gene families, as well as the disease genes for polycystic kidney disease and acute myelomonocytic leukemia. Several large-scale structural polymorphisms spanning hundreds of kilobasepairs were identified and result in gene content differences across humans. One of the unique features of chromosome 16 is its high level of segmental duplication, ranked among the highest of the human autosomes. While the segmental duplications are enriched in the relatively gene poor pericentromere of the p-arm, some are involved in recent gene duplication and conversion events which are likely to have had an impact on the evolution of primates and human disease susceptibility.

Chromosome 5 is one of the largest human chromosomes yet has one of the lowest gene densities. This is partially explained by numerous gene-poor regions that display a remarkable degree of noncoding and syntenic conservation with non-mammalian vertebrates, suggesting they are functionally constrained. In total, we compiled 177.7 million base pairs of highly accurate finished sequence containing 923 manually curated protein-encoding genes including the protocadherin and interleukin gene families and the first complete versions of each of the large chromosome 5 specific internal duplications. These duplications are very recent evolutionary events and play a likely mechanistic role, since deletions of these regions are the cause of debilitating disorders including spinal muscular atrophy (SMA).