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 (<10Kb) increases as the targeted fragment size increases. Our explanation for these quality changes is that as the targeted fragment size increases, the efficiency of the Cre joining two ends of the targeted fragments decreases. Therefore the complexity of the long pairs decreases. As a result of the PCR amplification, the redundancy of the targeted long pairs increases. On the other hand, the end joining of contaminated short fragments from the size selection gel is much more efficient than the targeted fragments. As the targeted fragment size increases, the population of the short pairs becomes more prominent. Third, we have also observed the chimeric rate increases with the targeted insert sizes. It is conceivable that as the efficiency of joining both ends of large fragments decreases, the chance of joining ends between DNA molecules increases, hence the increased chimeric rate. We have been testing several changes in the protocol. We now run two gels to size select targeted fragments to remove contaminated short fragments from the first gel. We have also tested the decrease of DNA concentration in the Cre recombination reaction to reduce the chance of inter-molecular joining. We will discuss the quality improvement of these changes.

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 (<10Kb) increases as the fragment size increases. Third, chimeric rate increases with the increasing fragment sizes. We have modified several steps to improve the quality of the long span PE libraries. The modification includes (1) the use of special PFGE program to reduce small fragment contamination; (2) the increase of DNA samples in the circularization step and prior to the PCR to reduce redundant reads; and (3) the decrease of fragment size in the double SPRI size selection to get a higher frequency of LoxP linker containing reads. With these modifications we have generated large gap size PE libraries with a much better quality.

As the demand of constructing Illumina libraries increases, we have started to modify the library construction protocol to adapt the use of multichannel pipette and 96-well plates. With the few simple modification steps, we have doubled the library production efficiency. These modifications include the shearing of DNA with Covaris E210, and the cleaning of enzymatic reactions and fragment size selection with SPRI beads and a magnetic plate holder. We have also designed a set of molecular barcodes to enable the sequencing of many libraries in parallel. The requirements of these barcodes include 4 bases, balanced GC, and at least 2 bases difference between barcodes. The barcode is attached to the adaptor so it does not require third sequencing primer and the barcoded library can be run on the same flowcell/run with other non-barcoded libraries. We have begun to assess the ability to assign reads and the potential bias towards certain barcodes after pooling different number of libraries. We have recently programmed the Biomek FX robot to carry out the library construction process. Although this process still require manual transfer of plates from robot to other work stations, the processing of 96 Illumina libraries takes approximately 6-8 hours. This semi-automated process represents a significant increase of library capacity comparing to the manual process. We will present the progress and the challenges of these scale-up processes.

Long-term climate change and periodic environmental extremes threaten food and fuel security¹ and global crop productivity^2-4. Although molecular and adaptive breeding strategies can buffer the effects of climatic stress and improve crop resilience⁵, these approaches require sufficient knowledge of the genes that underlie productivity and adaptation⁶-knowledge that has been limited to a small number of well-studied model systems. Here we present the assembly and annotation of the large and complex genome of the polyploid bioenergy crop switchgrass (Panicum virgatum). Analysis of biomass and survival among 732 resequenced genotypes, which were grown across 10 common gardens that span 1,800 km of latitude, jointly revealed extensive genomic evidence of climate adaptation. Climate-gene-biomass associations were abundant but varied considerably among deeply diverged gene pools. Furthermore, we found that gene flow accelerated climate adaptation during the postglacial colonization of northern habitats through introgression of alleles from a pre-adapted northern gene pool. The polyploid nature of switchgrass also enhanced adaptive potential through the fractionation of gene function, as there was an increased level of heritable genetic diversity on the nondominant subgenome. In addition to investigating patterns of climate adaptation, the genome resources and gene-trait associations developed here provide breeders with the necessary tools to increase switchgrass yield for the sustainable production of bioenergy.