Computational protocol: Variations in CCL3L gene cluster sequence and non-specific gene copy numbers

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Protocol publication

[…] We searched PubMed with the terms "MIP-1αP or MIP-1alpha P or LD78β or LD78beta or CCL3L1" for articles published in English up to the end of Jan 2010, and screened for publications specific to HIV/AIDS (and also separately for other diseases) and involving use of RT-PCR-based assays for examining copy numbers of CCL3L1 gene. Sequences of primers and probes used in RT-PCR assays in these publications were subjected to a nucleotide blast search against the human genomic plus transcript database on the National Center for Biotechnology Information (NCBI) website The sequences of genes containing these blast results were obtained from the UCSC Genomic Browser and aligned to the human reference sequence. Locations of exons, introns and untranslated regions of these genes were annotated from the NCBI Genome Browser. Pairwise, and multiple alignments were performed on these gene sequences via ClustalW in Molecular Evolutionary Genetics Analysis (MEGA 4) [] and in BioEdit []. Primers and probes were aligned individually with each gene and then with all pre-aligned genes in MEGA 4 and BioEdit.We used four different assays from the current literature (CCL3L_PP1, CCL3L_PP2, CCL3L_PP3, and CCL3L_PP4 in Table ) in a previously described subset of 47 European American and 48 African American healthy controls [] to quantify and examine the distribution of the copy numbers and the concordance rates between different assays in the two populations were assessed. Briefly, we used the specific primer/probe combinations (as shown in Table ) to quantify the copy numbers of CCL3L1 with the single-copy gene hemoglobin, beta (HBB) serving as the internal control. Real-time PCR was performed using an AB 7500 Fast System (Applied Biosystems Inc.). Cycling conditions were: 2 min at 50°C, 10 min at 95°C, and 40 cycles of 15 s at 92°C and 1 min at 60°C. Test genomic DNA samples was diluted to obtain a concentration of 2.5 ng/μl, and 2 ul (5 ng genomic DNA) was used in each reaction. We ran each sample in triplicates across three 96-well plates. We used the Applied Biosystems relative quantification program to determine gene copy numbers for the individual samples. The HBB gene that is present at two copies per diploid genome was used to standardize the gene copy number counts. Consistent with previous studies, we performed RT-PCR for each individual in triplicate and determined the normalized relative copy number by generating a standard curve and then normalizing across samples by the results of the HBB control gene and dividing the value obtained by the reference individual. Rounding off the outcome from the previous step to the nearest integer provided the estimated copy number counts. The copy numbers matching in triplicates and/or duplicates was used as the final copy number for an individual sample. […]

Pipeline specifications

Software tools Clustal W, MEGA, BioEdit
Application Genome data visualization
Organisms Human immunodeficiency virus 1, Cucumber necrosis virus, Homo sapiens