Additional file 1 of An analytical pipeline for identifying and mapping the integration sites of HIV and other retroviruses
Creators: name: Wells, Daria W.
name: Guo, Shuang
name: Shao, Wei
name: Bale, Michael J.
name: Coffin, John M.
name: Hughes, Stephen H.
name: Wu, Xiaolin
Publisher: name: figshare
Year: 2020
Subjects: subject: Genetics
subject: FOS: Biological sciences

description: Additional file 1: Table S1. Summary of Bioinformatics Analysis of Example Datasets. Table S2. Primers Used for the Patient Samples. Figure S1. The most frequent integration site motif in the raw data of Cohn et al. The unfiltered raw dataset [1] comprising 80 million sites, was searched using MEME to find the most common short sequence motif, which is shown in sequence logo format aligned with the sequence of the LTR1, which is the first (outside) PCR primer used by [1] to amplify integration sites. Figure S2. Likely mispriming sites in the Cohn et al. integration dataset. Sequences (from hg19) that were within 50 bases of the 6719 integration sites determined by [1] (provided by the authors) were searched using BLAST for matches to their LTR1 primer. 1114 such sequences (± 30 bases of the reported integration site; arrow and dashed line) were found; 10 are shown aligned with LTR1 and with the matching bases highlighted in yellow. The chromosomal location of each site is shown to the left, with the number of patients reported to have a provirus at that site shown in parentheses. Figure S3. Plausible mechanisms for the erroneous identification of integration sites. The raw data used by [1] (NCBI accession number SRP045822) were searched for reads containing the cellular sequence 3′ of the reported integration site. No correct integration events were found, but several types of aberrant sequences were identified, including the 3 examples shown and labeled “raw read.” A. Double mispriming and PCR recombination. This sequence was most likely created by mispriming of LTR1 on the matching sequence (yellow) on chromosome 11 (magenta) as well as by LTR2 (green), and mispriming on a matching sequence on chromosome X (green), followed by recombination during PCR across the 8-base match indicated. B. Mispriming by a perfect fusion of sequences of LTR2-LTR1 on the same chromosome 11 sequence. C. Apparent correct integration (LTR2 followed by the 3′ 7 bases of the HIV-1 LTR1) two bases upstream of the reported integration site. The boxed sequence shows the 3′ most 7 nucleotides of the LTR, which are not in LTR2, but must be present in every correctly amplified integration site. Figure S4. The most common DNA sequence motif in the integration site datasets. Sequences ±50 base pairs of the reported integration sites from various studies are shown [1, 6, 11, 22], and 10,000 randomly chosen hg19 sequences and 10,000 Alu sequences were searched for common motifs using MEME [23]. The top hit in each case was aligned to the “integration site motif” of [1] (see text). The arrow above the sequence marks the site reported to be the preferred integration site [which is one nucleotide away from the site found at this site in the raw reads from patient 3, time point 3 (Figure S3). Figure S5. Sequence motifs adjacent to the integration site in various datasets. The patterns of preferred nucleotides in the host DNA immediately adjacent to the integration sites in patients reported by Maldarelli et al. [6] and Cohn et al. [1] were compared to data from cells infected in vitro (a PBMC dataset and a HeLa dataset). The sequence motifs were determined as previously described [8]. In the in vitro datasets, the target site nucleotides form a weak palindrome that matches what has been previously determined for HIV-1 using much smaller datasets [7, 8]. The preferred nucleotides in the Maldarelli dataset also match this motif. However, while the data of Cohn et al. shows some evidence of the palindrome; the sequence is weak and is not entirely symmetrical. The sequence from the Patient 3.3 sample (third time point) is obviously quite different from all the other data. Figure S6. Matching the Alu “integration sites” reported for the Patient 3.3 sample to the Alu consensus. The Alu consensus sequence is shown at the bottom. The quality of the match to the consensus is shown is the graph. The break in the match suggests the point in the sequence at which recombination frequently occurred.