title: Clostridium thermocellum 1313 resequencing
design: Illumina TruSeq libraries were prepared as described in the manufactures methods (Part# 15005180 RevA)  following the low throughput protocol.  In short, 3 ug of DNA was sheared to a size between ~200 bp and 1000bp by nebulization (nitrogen) for 1 min at 30psi.  The sheared DNA was purified on a Qiagen Qiaquick Spin column (Qiagen).  The sheared material was accessed for quantity with a Qubit broad range double stranded DNA assay (Life Technologies) and quality by visualization on an Agilent Bioanalyzer DNA 7500 chip (Agilent).  One microgram of sheared DNA was used for library preparation following the manufactures method.   Steps included end repair followed by an AMPure XP bead clean up, adenylation of 3’ ends and adapter ligation followed by a second AMPure XP bead clean up, purification of the library by gel selection, DNA enrichment with 10 cycles of amplification and a final AMPure XP bead clean up.  Libraries were validated by Qubit (Life Technologies) and visualized by Agilent Bioanalyzer for appearance and size determination.  Samples were normalized using the Illumina’s Library dilution calculator to a 10nM stock, and subsequently run on an Illuimna MiSeq Instrument. <P>     From Simultaneous achievement of high ethanol yield and titer in Clostridium thermocellum  Liang Tian†, Beth Papanek†, Daniel G. Olson, Thomas Rydzak, Evert K. Holwerda, Tianyong Zheng, Jilai Zhou, Marybeth Maloney, Nannan Jiang, Richard J. Giannone, Robert L. Hettich, Adam M. Guss and Lee R. Lynd      Biotechnology for Biofuels20169:116  DOI: 10.1186/s13068-016-0528-8©  The Author(s) 2016  Received: 3 March 2016Accepted: 19 May 2016Published: 2 June 2016      C. thermocellum strain AG553 grows much more slowly than wild type [Papanek B, Biswas R, Rydzak T, Guss AM: Characterization of Clostridium thermocellum strains with disrupted fermentation end-product pathways. Metab Eng 2015, 32:49–54], presumably due to metabolic bottlenecks or imbalances. To improve the growth rate, the strain was serially transferred approximately daily in rich medium (CTFUD) with 5 g/L cellobiose such that faster growing cells would begin to dominate the culture. Each transfer consisted of a ~1000-fold dilution, or approximately 10 generations. After 150 transfers (~1500 generations), the increase in ethanol production had slowed (Figure 2A) and growth rate had significantly improved (Table 1).     The nature of this type of laboratory evolution results in the cultures being a mixture of genetic backgrounds, as different cells within the population acquire different mutations. This diversity of genotypes makes understanding of the causative mutation difficult and further genetic manipulation impossible. Therefore, the isolation of an individual genetic background strain was necessary. An individual member of the population was isolated via single colony purification, cells were prepared and DNA was isolated at time = 0 and designated Q0. DNA from An individual member of the population was isolated via single colony purification, cells were prepared and DNA was isolated for lineage Q1 after 20, 30, 90 and 150 transfers.  An individual member of the population was isolated via single colony purification, cells were prepared and DNA was isolated for lineage Q2 after 30 and 150 transfers.  The raw read files are presented for      T=0 (Ctherm1313ReSeqQ0_S1_L001_R1_001.fastq.gz &Ctherm1313ReSeqQ0_S1_L001_R2_001.fastq.gz)  Lineage Q1 20 transfers (Ctherm1313ReSeqQ1-20_S2_L001_R1_001.fastq.gz & Ctherm1313ReSeqQ1-20_S2_L001_R2_001.fastq.gz)  Lineage Q1 30 transfers (Ctherm1313ReSeqQ1-30_S3_L001_R1_001.fastq.gz & Ctherm1313ReSeqQ1-30_S3_L001_R2_001.fastq.gz)  Lineage Q1 90 transfers (Ctherm1313ReSeqQ1-90_S4_L001_R1_001.fastq.gz & Ctherm1313ReSeqQ1-90_S4_L001_R2_001.fastq.gz)  Lineage Q1 150 transfers (Ctherm1313ReSeqQ1-150_S5_L001_R1_001.fastq.gz & Ctherm1313ReSeqQ1-150_S5_L001_R2_001.fastq.gz)  Lineage Q2 30 transfers (Ctherm1313ReSeqQ2-30_S6_L001_R1_001.fastq.gz & Ctherm1313ReSeqQ2-30_S6_L001_R2_001.fastq.gz)  Lineage Q2 150 transfers (Ctherm1313ReSeqQ2-150_S7_L001_R1_001.fastq.gz & Ctherm1313ReSeqQ2-150_S7_L001_R2_001.fastq.gz)