DNA sequencing is performed in both the forward and reverse directions so that the complementary strands can be compared to one another for quality control purposes. If it is not possible to get sequence from both strands, for example following a C-stretch, then the same strand can be sequenced twice in separate reactions. The goal is to have at least double coverage of every nucleotide being assessed either through sequencing the top and bottom strand or sequencing the same strand twice.
The sequencing process does not always lead to beautiful data that is unambiguous for each base. Some regions, such as the C-stretches, are challenging to decipher and may not even be included in the final interpretation (Stewart et al. 2001). Sequencing chemistries and instruments have improved in recent years leading to more even peaks, better sensitivity and less noise. However, experienced analysts must still manually review each nucleotide with the aid of computer software and then edit base calls when the base-calling algorithm has made an incorrect call. At present there is no publicly available software that can robustly evaluate mtDNA sequence data in a reliable and automated fashion without manual intervention.
The sequence editing process is aided by alignments from the multiple sequences generated over a region for the same sample. Computer programs such as Sequencer (GeneCodes, Ann Arbor, MI) align the forward and reverse sequencing reactions and allow the sequencing electropherograms for each reaction to be evaluated side-by-side. For casework samples that utilize smaller PCR products there is overlap between them (see Figure 10.3) that permits a further measure of quality assurance in the final compiled sequence. In addition, two forensic analysts must independently examine, interpret, and edit sequence matching results as a final quality assurance measure (Isenberg 2004).
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This book discusses the futility of curing stammering by common means. It traces various attempts at curing stammering in the past and how wasteful these attempt were, until he discovered a simple program to cure it. The book presents the life of Benjamin Nathaniel Bogue and his struggles with the handicap. Bogue devotes a great deal of text to explain the handicap of stammering, its effects on the body and psychology of the sufferer, and its cure.