Retinopathy-positive cerebral malaria patient
DBLα tags were PCR-amplified from cDNA from a selection of patients with severe brain swelling as well as patients with high CIDRα1.7 transcription. 20-40 cycle PCR amplification was done in either one step, using the previously described varF_dg2 and brlong2 primers (Lavstsen et al., 2012) ligated to 5’ and 3’ MiSeq adaptor sequences (5’-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGGCAMGMAGTTTYGCNGATATWGG and 5’-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGTCTTCDSYCCATTCVTCRAACCA) or in 2 steps with varF_dg2 and brlong2 primers first, followed by MiSeq adaptor-DBLα primers (sequences shown above). PCR conditions were as follows: 98°C for 45 s, followed by 20-40 cycles of 98°C for 10 s, 50°C for 20 s, 68°C for 20 s and final extension at 72°C for 7 min. Amplicons were gel-purified and served as the template for second-round amplification where P5 and P7 index sequences were added in 10 cycles using Nextera primers (Illumina) and the aforementioned PCR conditions. Amplicons were purified using FlashGels (Lonza) and quantified using the KAPA library quantification kit (Kapa Biosystems) by qPCR performed on an Applied Biosystems 7500 Fast real-time PCR machine (Applied Biosystems). Libraries were combined at 500 pM and denatured using 0.2N NaOH according to the Illumina MiSeq protocol. The denatured library was diluted in HT1 hybridization buffer (Illumina) to 10 pM, and a 12.5 pM PhiX Control library added to a final concentration of 40%. The library was loaded into a 600-cycle V3 cartridge (Illumina) for sequencing.