Pool-Seq on cercariae of S.mansoni SmBRE from sympatric snail hosts
The Illumina TruSeq DNA sample preparation kit (FC-121-2001, Illumina Inc., San Diego, USA) was used according to the manufacturer's six-steps protocol: 1) 1 µg of DNA was sonicated for 20 minutes by using a Bioruptor (power set on “High”, cycles of 30 seconds “ON” and 30 seconds “OFF”); 2) Extremities of sonicated DNA fragments were repaired ; 3) an A-base was added to these extremities ; 4) Universal (5'-AAT¬GATACGGCGACCACCGAGATCTA¬CACTCTTTCCCTACACGACGCTCTTCCGATCT-3’) and strain-specific (C: 5’-GATCGGAAGAGCACACGTCTGAACTCCAGTCACTGAC¬CAATCTCGTATGCCGTCTTCTGCTTG-3’ ; IC: 5’-GATCGGAAGAGCA¬CACGTCTGAACTCCAGTCACCGATGTATCTCGTATGCCGTCTTCTGCTTG-3’) adapters were then ligated to these extremities ; 5) DNA products were migrated on 2% agarose gel and 500bp +/-25 fragments were purified (DNA insert size of 380 bp +/-25); 6) 12 cycles of PCR amplification were performed to select only DNA fragments carrying both universal and strand specific adapters; 6) Libraries were then validated and quantified on a DNA1000 chip on a Bioanalyzer (Agilent) to determine size and concentration. The cluster generation process was performed on cBot (Illumina Inc.) by using the Illumina Paired-End DNA sample preparation kit (FC-102-1001, Illumina Inc.). DNA libraries were denatured using 0.1 N NaOH then diluted to 8 pM. Diluted libraries were first hybridized to the flow cell (one library per lane) and then amplified leading to DNA cluster formation. After DNA linearization and free-extremity blocking, the first sequencing primer was hybridized to the free-extremity of DNA fragments.