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Protocol publication

[…] scribed (). To confirm amplification, PCR products were run on 1% agarose gel with 240 V, 300 mA for 1.5 h, and bands were visualized under UV light. Amplicons were then combined into one pool (5 µl of amplicon 1 and 10 µl each of amplicons 2, 3, and 4) and ultra-deep sequenced at the Wellcome Trust Sanger Institute (Cambridge, UK)., Libraries were prepared from 50–1,000 ng DNA as described previously (, ) using one of 96 multiplex adaptors for each pool of amplicons. Paired-end sequencing with a read length of 300 bp was performed using the Illumina MiSeq instrument as described previously (). Consensus sequences were generated by de novo assembly (i.e., without a reference sequence) using Iterative Virus Assembler ()., Gaps in the de novo assembled genomes were filled using maternal or a reference sequence. The reads were then mapped to the assemblies using MOSAIK () with default parameters. A subtype C reference genome was annotated with the location of the epitopes of interest. The de novo assemblies were aligned using MUSCLE () to the annotated reference to determine the location of the epitopes in each assembly. V-Phaser 2 with default parameters was used to call variants () and V-Profiler to call haplotypes () for the HLA-associated epitopes. Only haplotypes with greater than 1% frequency were included in the analysis., HIV genomes from 11 PSP mother–child pairs were ultra-deep sequenced. Maternal time points included closest to delivery, if available, and 5–7 yr after delivery, if available; children’s time points included earliest available, ∼2.5, ∼5, and ∼7 yr of age or only the earliest and 5 yr of age time points (43 full genomes and seven partial genomes were generated). To confirm the close relat […]

Pipeline specifications

Software tools IVA, MOSAIK, MUSCLE