北京大学生物信息学-第三周-序列数据库 BLAST

序列数据库

• Genbank是美国国家生物技术信息中心(National Center for Biotechnology Information ，NCBI)建立的DNA序列数据库，从公共资源中获取序列数据
• SRA（Sequence ReadArchive）数据库是用于存储二代测序的原始数据，包括 454，Illumina，SOLiD，IonTorrent，Helicos 和 CompleteGenomics。除了原始序列数据外，SRA现在也存在raw reads在参考基因的比对信息。

BLAST算法初探

BLAST Ideas: Seeding‐and‐extending：种子-扩展

• Find matches (seed) between the query and subject；查找查询序列和目标序列之间的匹配（种子）
• Extend seed into High Scoring Segment Pairs (HSPs)；将种子扩展成高分段对（HSPs）
  – Run Smith‐Waterman algorithm on the specified region only.特定区域
• Assess the reliability of the alignment.评估校准的可靠性
  
  Seeding：
  For a given word length w (usually 3 for proteins and 11 for
  nucleotides), slicing the query sequence into multiple
  对于给定的单词长度w（通常蛋白质用3和核苷酸用11），将查询序列切片成多个。
  continuous “seed words” 种子单字
  Speedup: Index database 索引数据库-加速
  The database was pre‐indexed to quickly locate all positions in the database for a given seed.
  数据库被预索引以快速定位数据库中给定种子的所有位置。
  
  E‐Value: How a match is likely to arise by chance：匹配偶然发生的可能
  • The expected number of alignments with a given score that would be expected to occur at random in the database that has been searched
  在已搜索的数据库中，与预期的给定分数对齐的预期数量
  – e.g. if E=10, 10 matches with scores this high are expected to be found by chance
  例如，如果E=10，期望随机找到与该分数相匹配的10个匹配项
  

BLAST详解

Why BLAST？

• "Homology is the central concept for all of biology.”
  同源性是所有生物学的中心概念。
• BLAST is the tool most frequently used for calculating sequence similarity, by searching the databases.
  BLAST是通过搜索数据库来计算序列相似性的最常用工具。
• If you work with one or a few proteins or genes, it can tell you about their conservation, active sites, structure and regulation in other organisms, etc.
  如果你研究一个或几个蛋白质或基因，它可以告诉你它们在其他有机体中的保存、活性位点、结构和调控等。
  What BLAST does?
• Identity: the occurrence of exactly the same nucleotide or amino acid in the same position in aligned sequences.
  完全相同的核苷酸或氨基酸在同一排列顺序的相同位置上的出现。
• Similarity: measure the sameness or difference of the sequences
  度量序列的相同点或不同点
• Homology: is defined in terms of shared ancestors. Homologous sequences are often similar. Sequence regions that are homologous are also called conserved regions.
  定义在共同的祖先。同源序列通常是相似的。
  动态规划：准确度高，速度不快
  FASTA,BLAST：不完全准确，但速度快
  

0 Filtering 过滤

• To prevent the production of large numbers of statistically significant but biologically uninteresting results.
  为了防止产生大量在统计上有意义，但在生物学生无趣的结果。

• Low complexity and repeats 低复杂性和重复性, i.e.

• (CA)n

• KLKLKLKLKLKL

• Cover these regions with the following letters 用下列字母覆盖这些区域

• Ns (for nucleotide residues) 核苷酸残余

• Xs(for amino acid residues) 氨基酸残余

• -Fflag: filter query sequence 过滤查询序列

1 Seeding 播种

• Make a w-letter word list of the query sequence
  • Usually 3 for amino acid sequences, and
  • 11 for nucleotide sequences
• For a query sequence with n letters, the number of words is n –w + 1
• -W flag: word size
  

2 Search word hits 搜索词命中率

• Scoring matrix
  • for amino acids, BLOSUMor PAM
  • For DNA words, a match is scored as +5 and a mismatch as -4, or as +2 and -3
• No gaps are allowed
• The words whose scores are greater than the thresholdTwill remain in the possible matching words list
  得分大于阈值的单词将保留在可能匹配的单词列表中
  

3 Scanning 扫描

• HashTable: direct addressing method 直接寻址方法
• Deterministic finite automaton/finite state machine: much faster
  确定性有限自动机/有限状态机：更快
  

4 Extending->HSP 扩展

• Cutoff score S
  

5 Significance evaluation 重要评估

• Raw scores: have little meaning without detailed knowledge of the scoring system used.
  原始分数：如果不详细了解所使用的评分系统，就没有什么意义。
• Bit scores: normalizing a raw score using the formula.
  位分数：使用公式对原始分数进行规范化。
  
• E values: corresponding to a given bit score
  对应于给定的位分数