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Recently, the Cas9-CRISPR system has proven to be a useful tool for genome editing in eukaryotes, which repair the double stranded breaks made by Cas9 with non-homologous end joining or homologous recombination. Escherichia coli lacks non-homologous end joining and has a very low homologous recombination rate, effectively rendering targeted Cas9 activity lethal. We have developed a heat curable, serializable, plasmid based system for selectionless Cas9 editing in arbitrary E. coli strains that uses synthetic CRISPRs for targeting and -red to effect repairs of double stranded breaks. We have demonstrated insertions, substitutions, and multi-target deletions with our system, which we have tested in several strains.

Paired-end library sequencing has been proven useful in scaffold construction during de novo whole genome shotgun assembly. The ability of generating mate pairs with > 8 Kb insert sizes is especially important for genomes containing long repeats. To make mate paired libraries for next generation sequencing, DNA fragments need to be circularized to bring the ends together. There are several methods that can be used for DNA circulation, namely ligation, hybridization and Cre-LoxP recombination. With higher circularization efficiency with large insert DNA fragments, Cre-LoxP recombination method generally has been used for constructing >8 kb insert size paired-end libraries. Second fragmentation step is also crucial for maintaining high library complexity and uniform genome coverage. Here we will describe the following three fragmentation methods: restriction enzyme digestion, random shearing and nick translation. We will present the comparison results for these three methods. Our data showed that all three methods are able to generate paired-end libraries with greater than 20 kb insert. Advantages and disadvantages of these three methods will be discussed as well.

Fosmid or BAC end sequencing plays an important role in de novo assembly of large genomes like fungi and plants. However construction and Sanger sequencing of fosmid or BAC libraries are laborious and costly. The current 454 Paired-End (PE) Library and Illumina Jumping Library construction protocols are limited with the gap sizes of approximately 20 kb and 5 kb, respectively. In the attempt to understand the limitations of constructing PE libraries with greater than 30Kb gaps, we have purified 18, 28, 45, and 65Kb sheared DNA fragments from yeast and circularized the ends using the Cre-loxP approach described in the 454 PE Library protocol. With the increasing fragment sizes, we found a general trend of decreasing library quality in several areas. First, redundant reads and reads containing multiple loxP linkers increase when the average fragment size increases. Second, the contamination of short distance pairs (

Long insert size paired-end (PE) sequences play a key role in de novo assemblies of complex genomes. There is no reliable method, however, for generating 30Kb or longer insert size PE libraries that is suitable for 454 or Illumina sequencing. In the attempt to understand the limitations of constructing greater than 30Kb PE libraries, we have purified 18, 28, 45, and 65Kb sheared DNA fragments from yeast and circularized the ends using the Cre-loxP approach described in the 454 Titanium long paired-end library protocol. With the increasing insert sizes, we found a general trend of decreasing library quality in several areas. First, the portion of redundant pairs increases from 18percent to almost 40percent of the total pairs when the average fragment size increases from 18Kb to 45Kb. Second, the contamination of short insert size pairs (

Paired-end library sequencing has been proven useful in scaffold formation during the de novo assembly of genomic sequences. The current protocol for constructing 454 Titanium paired-end libraries (Roche/454 unpublished) has allowed a routine production of libraries with 3 to 20 Kb insert sizes, which is suitable for assembling large eukaryote genomes with large segments of repeats. This protocol however was designed for manual operation that handles individual tubes, which is laborious and low in throughput. To increase the productivity, we modified the procedure by adopting an 8-strip tube, multiple channel pipette, and 96 magnetic O-ring plate. Other modifications include using the Promega Wizard SV 96-well column instead of gel electroelution after gel separation; replacing nebulization with sonication; and applying AMPure-Beads in certain steps for DNA purification. These changes could increase the productivity of our manual library construction process to 24 libraries a week. These modifications also make it possible for the future development of automatic or semi-automatic procedures. Furthermore, we have also modified some of the steps to improve the library quality and productivity. These steps include applying UV cross linked DNA as carrier DNA and using barcoded adapters in library construction for sequencing pooled libraries.

Paired-end library sequencing has been proven useful in scaffold construction during de novo assembly of genomic sequences. The ability of generating mate pairs with 8 Kb or greater insert sizes is especially important for genomes containing long repeats. While the current 454 GS LT Paired-end library preparation protocol can successfully construct libraries with 3 Kb insert size, it fails to generate longer insert sizes because the protocol is optimized to purify shorter fragments. We have made several changes in the protocol in order to increase the fragment length. These changes include the use of Promega column to increase the yield of large size DNA fragments, two gel purification steps to remove contaminated short fragments, and a large reaction volume in the circularization step to decrease the formation of chimeras. We have also made additional changes in the protocol to increase the overall quality of the libraries. The quality of the libraries are measured by a set of metrics, which include levels of redundant reads, linker positive, linker negative, half linker reads, and driver DNA contamination, and read length distribution, were used to measure the primary quality of these libraries. We have also assessed the quality of the resulted mate pairs including levels of chimera, distribution of insert sizes, and genome coverage after the assemblies are completed. Our data indicated that all these changes have improved the quality of the longer insert size libraries.

Paired-end library sequencing has been proven useful in scaffold construction during de novo assembly of genomic sequences. The ability of generating mate pairs with 8 Kb or greater insert sizes is especially important for genomes containing long repeats. While the current 454 GS LT Paired-end library preparation protocol can successfully construct libraries with 3 Kb insert size, it fails to generate longer insert sizes because the protocol is optimized to purify shorter fragments. We have made several changes in the protocol in order to increase the fragment length. These changes include the use of Promega column to increase the yield of large size DNA fragments, two gel purification steps to remove contaminated short fragments, and a large reaction volume in the circularization step to decrease the formation of chimeras. We have also made additional changes in the protocol to increase the overall quality of the libraries. The quality of the libraries are measured by a set of metrics, which include levels of redundant reads, linker positive, linker negative, half linker reads, and driver DNA contamination, and read length distribution, were used to measure the primary quality of these libraries. We have also assessed the quality of the resulted mate pairs including levels of chimera, distribution of insert sizes, and genome coverage after the assemblies are completed. Our data indicated that all these changes have improved the quality of the longer insert size libraries.

As Sanger sequencing is being replaced by higher throughput and lower cost of next generation sequencing, finishing microbial genomes will face two major challenges. First, the technology will need to be fast enough to handle many more drafted genomes. Second, it will have to incorporate a clone-free approach to fill gaps. We have been testing a method that utilizes a universal "bubble-tag" to perform primer walking and gap closure in a clone-free condition. The ?bubble-tag? method was first described by Doug Smith (PCR Methods Appl. 2: 21-27, 1992) to amplify and sequence lambda DNA. There is no evidence however, that this approach will work for the more complex microbial genome. Here we describe the experimentation of this approach in primer walking of the E. coli genome. Genomic DNA was sheared, blunt-end repaired, or digested with frequent cutters, and ligated to bubble adaptors. Site specific primers were used together with the universal bubble primer to amplify and sequence the regions of interest. Different primer concentration was tested in order to generate long amplified templates. This approach enables primer walking and gap filling in a clone-free draft sequencing process. More importantly, the uniformity of this approach is amenable for an automated finishing process.

We have constructed and sequenced several 454 paired end LT libraries with 3 Kb insert size to assess the quality and effectiveness of the technology in the whole genome assembly. A set of quality metrics, which include levels of redundant reads, linker positive, linker negative, half linker reads, and driver DNA contamination, and read length distribution, were used to measure the primary quality of these libraries. We have also assessed the quality of the resulted mate pairs including levels of chimera, distribution of insert sizes, and genome coverages after the assemblies are completed. In general, the technology is performing as expected with 3 Kb fragments. We have made several modifications in the procedure to improve the quality of the libraries, especially in the areas of reducing redundant reads and eliminating pUC driver DNA contamination. We have also compared two assembly tools for their performance in the assembly of mixed Sanger and 454 shotgun or paired-end data. We will report the quality of these libraries and the comparison of the assemblies with shotgun and paired-end reads. The current GS paired end LT library construction protocol would not work for fragment sizes larger than 3 Kb. The major obstacles are the low recovery of large DNA fragments after multiple purification steps and the lack of an efficient way to circularize large DNA fragments. We are in the process of modifying some steps to overcome these obstacles. We will present results of the improvements.

Fosmid Ditags as a New Technology Developed at JGIZe Peng1, Ilya Malinov1, Doug Smith1, Feng Chen1, Paul Richardson1, Len A. Pennacchio1, and Jan-Fang Cheng11Lawrence Livermore National LaboratoryUS Department of Energy Joint Genome Institute, Walnut Creek, CAPaired end reads from large insert DNA libraries are essential for detecting chromosome rearrangements as well as connecting sequence scaffolds of draft genomes. However, fosmid and BAC end sequencing remains challenging as well as expensive. Ditag sequencing of fosmid ends represents a cost effective way to generate paired end sequences from large genomic fragments. We present results from several ditag libraries from human, fungi, and bacteria, which were sequenced using 454 technology. Several software tools were developed to analyze the resulted ditag sequences. These tools have been used to (1) create suffix arrays of the reference genomes; (2) filter, trim, and prepare the paired 18mer ditag sequences for analysis; (3) search for 18mer strings for matches; and (4) score the chromosome locations of ditag pairs. For testing the accuracy and sensitivity of detecting chromosome rearrangements, we have generated 235,394 unique ditag pairs from a breast cancer genome. These fosmid sequence tags represent about 3.1-fold clone coverage of the genome. We have identified 59 rearrangements including 13 translocations, 23 deletions, and 23 inversions. Of those, 14 have been previously detected by an independent approach of using the BAC end sequence profiling data. We are in the process of generating ditags from a green microalga (Micromonas pusilla NOUM17), a deuteromycete fungus (Trichoderma virens Gv29-8), a poplar rust (Melampsora larici-populina 98AG31), and 3 prokaryotes. So far we have found that most ditag pairs could be localized to the draft genomes with the predicted distances and some ditags helped connecting sequence scaffolds to improve the continuity of the assemblies. The ditag technology in conjunction with the 454 sequencing provides a high throughput approach to assist shotgun sequence assemblies and characterize cancer genomes.This work was performed under the auspices of the US Department of Energy's Office of Science, Biological and Environmental Research Program, and by the University of California, Lawrence Livermore National Laboratory under Contract No. W-7405-Eng-48, Lawrence Berkeley National Laboratory under contract No. DE-AC02-05CH11231 and Los Alamos National Laboratory under contract No. DE-AC02-06NA25396.

Genetic studies in non-human primates serve as a potential strategy for identifying genomic intervals where polymorphisms impact upon human disease-related phenotypes. It remains unclear, however, whether independently arising polymorphisms in orthologous regions of non-human primates leads to similar variation in a quantitative trait found in both species. To explore this paradigm, we studied a baboon apolipoprotein gene cluster (APOA1/C3/A4/A5) for which the human gene orthologs have well established roles in influencing plasma HDL-cholesterol and triglyceride concentrations. Our extensive polymorphism analysis of this 68 kb gene cluster in 96 pedigreed baboons identified several haplotype blocks each with limited diversity, consistent with haplotype findings in humans. To determine whether baboons, like humans, also have particular haplotypes associated with lipid phenotypes, we genotyped 634 well characterized baboons using 16 haplotype tagging SNPs. Genetic analysis of single SNPs, as well as haplotypes, revealed an association of APOA5 and APOC3 variants with HDL cholesterol and triglyceride concentrations, respectively. Thus, independent variation in orthologous genomic intervals does associate with similar quantitative lipid traits in both species, supporting the possibility of uncovering human QTL genes in a highly controlled non-human primate model.