title: Target:IS:AC:F1
design: Human T-cell leukemia virus type-1 (HTLV-1) is the causative agent of adult T-cell leukemia (ATL). We took advantage of next-generation sequencing technology, a tag system, and an in-silico analysis pipeline to develop and internally validate a new high-throughput methodology. Our method enables accurate monitoring of clonal expansion of HTLV-1 infected cells based on integration sites of provirus and measure the size of infected clones. The method was proved to accurately measure the size of clones by analyzing control samples with already known clone sizes and clinical samples (Firouzi et al. Genome Medicine). Taking advantage of our new method to detect clonality, we further analyzed a greater number of samples from asymptomatic carriers (ACs) and different the subtypes of ATL (Farmanbar et al. BMC Medical Genomics). Our clonality analysis method included two main aspects: (1) wet experiments, and (2) in silico analysis. We have provided details of the method design, optimized experiment protocols, and in-silico data processing workflow in (Firouzi et al. Genome Medicine) and (Farmanbar et al. BMC Medical Genomics). We employed a library preparation protocol specifically designed to isolate HTLV-1 integration sites. The final products in the library that we generated contained all the specific sequences necessary for the Illumina HiSeq 2000/2500 platform. Some of the samples contain spike-in integration sites.These products included a 5'-flow cell binding sequence, a region compatible with read-1 sequencing primer, 5-bp random nucleotides, 5-bp known barcodes for multiplexing samples, HTLV-1 long terminal repeat (LTR), human or HTLV-1 genomic DNA, a region compatible with read-2 and read-3 sequencing primers, 7 or 8-bp random tags, and a 3'-flow cell binding sequence from 5' to 3', respectively. Tags acts as molecular barcodes which give DNA fragment unique signatures before PCR. Information on the frequency of observed tags from the NGS data was used to remove PCR duplicates and thereby estimate the original clonal abundance in the starting sample (Firouzi et al. Genome Medicine) and (Farmanbar et al. BMC Medical Genomics). In the current project (Farmanbar et al. BMC Medical Genomics), in total, eighteen clinical samples were obtained from Joint Study on Predisposing Factors of ATL Development (JSPFAD) biomaterials bank of HTLV-1 carriers. We categorized clones into four size groups, “very small”, “small”, “big”, and “very big”, based on the patterns of clonal growth and observed clone sizes. We propose an empirical formal model based on deterministic finite state automata (DFA) analysis of real clinical samples to illustrate patterns of clonal expansion (Farmanbar et al. BMC Medical Genomics). Please contact Sanaz Firouzi (Ph.D) (firouzisanaz1@gmail.com) for further information about details of samples and analysis.