Large construction kit

Based on functional screening, a total of 173 positive fosmid clones were randomly selected from variant levels of halo against screening substrates for full sequencing (Figure S1), including 68 endoglucanase-, 15 exoglucanase-, 40 β-glucosidase-, and 50 endoxylanase-positive clones (10 out of these clones exhibited dual enzyme specificities). After cell density normalization, every 10 to 12 different fosmids were pooled as a sample for the whole fosmid DNA extraction using the QIAGEN Large-Construction kit according to manufacturer’s instructions. Extracted DNA was directly subjected to 454 pyrosequencing (Roche Diagnostics, USA). After assembly (Newbler 2.5.3), coding regions and bacterial operons were predicted by FGENESB (http://linux1.softberry.com/berry.phtml). The predicted amino acid sequences were aligned to the eggNOG v4.5.1 database [35 (link)] and KEGG for further functional prediction (E-value <1.0e⁻⁵). Searches for carbohydrate-active enzymes was performed against dbCAN database with default parameters. Signal peptides were predicted by SignalP 4.1 in CBS (http://www.cbs.dtu.dk/services/SignalP/). Molecular masses and isoelectric points were predicted by ExPASy (http://www.expasy.ch/tools/protparam.html). Protein cellular location was predicted using CELLO v.2.5 (http://cello.life.nctu.edu.tw) [36 (link)].

Four-micrometer deparaffinised section was incubated in pre-treatment solution (Abbott Molecular, Downers Grove, IL) at 80 °C for 40 min and then in protease solution (Abbott Molecular) for 40 min at 37 °C. Fibroblast growth factor receptor 1 (FGFR1) FISH was performed with locus-specific bacterial artificial chromosome (BAC), RP11-100B16 (chr8:38,358,839-38,522,417). We obtained the BAC clone from Invitrogen (Carlsbad, CA) and purified it with a large construction kit (Qiagen, Valencia, CA). DNA from the BAC clone was labelled with SpectrumOrange using a nick translation kit (Abbott Molecular). FGFR1 BAC probe and FGFR2 probe (Vysis LSI FGFR2 SpectrumOrange Probe, 08N42-020, Abbott Molecular) were denatured at 73 °C for 5 min and hybridised at 37 °C for 20 h. Post-hybridisation washes were performed according to the protocol supplemented. Slides were mounted in 4′,6-diamidino-2-phenylindole/anti-fade and viewed with a fluorescence microscope. The FGFR1 or FGFR2 was considered to be amplified if the average gene copy number was ≥6, and high-level amplification was defined as an average gene copy number ≥10.