Similar protocols

Pipeline publication

[…] ystem (Promega) according to the manufacturer's instructions. The constructs generated were used to transform DH5α Escherichia coli competent cells. Thirteen to twenty white colonies were screened by colony PCR for each of the 31 samples for sequence analysis. Plasmid inserts were amplified using pGEM®-T-specific primers T7 and SP6 and subjected to double stranded sequence analysis with dye-labeled dideoxy terminators with both T7 and SP6 primers (genetic analyzer 3500xL, Applied Biosystems®, Life Technologies, USA). The core sequences from the two sampling time points selected (or only baseline in the case of the nonresponder patient E or responder patients) were edited and assembled using Sequencher software v.5.0.1 (Gene Codes Corporation, Ann Arbor, MI, USA)., Though PCR mutations are a well-known problem, our pipeline for clonal analysis included amplifications that were performed using sufficient molecules as template in order to minimize the error rate due to PCR., Sequence alignments were obtained with the MAFFT program implementing iterative refinement, weighted sum-of-pair and scores, and a consistency score obtained from local alignments []., Phylogenetic trees were inferred by FastTree-2 [], under the GTR + CAT model. FastTree automatically scales the tree searching effort proportionally to the number of sequences in the dataset [i.e., the program performs up to 4 × log⁡2(N) rounds of minimum-evolution nearest neighbor interchange (NNI), 2 rounds of subtree pruning regrafting (SPR) moves, and up to 2 × log⁡(N) rounds of maximum likelihood NNIs, where N is the number of unique sequences in the dataset]. We set the program to start from an exhaustive-search BIONJ tree and turned off top-hit heuristics to tree improve searches. Bootstrapping was used to both obtain branch supports and cope with phylogenetic uncertainty in evolutionary analyses. To this aim, we generated 1000 bootstrapped datasets using Seqbo […]

Pipeline specifications

Software tools Sequencher, MAFFT, FastTree