上机实习四：BLAST序列相似性搜索工具的使用.pptVIP

上机实习四：基本局部比对搜索工具blast的使用  * Step 1: Choose your sequence Sequence can be input in FASTA format or as accession number BLAST搜索的4个步骤 ①选择你所感兴趣的序列号或序列，将它粘贴到BLAST的输入框中。 ②选择一个BLAST程序(blastp，blastn，blastx，tblastx，tblastn)。 ③选择一个用于搜索的数据库。一个通常的选择是去冗余 (nr)。 ④为搜索和输出格式选择可选参数。 Example of the FASTA format for a BLAST query Step 2: Choose the BLAST program Step 2: Choose the BLAST program blastn (nucleotide BLAST) blastp (protein BLAST) tblastn (translated BLAST) blastx (translated BLAST) tblastx (translated BLAST) Step 3: choose the database nr = non-redundant (most general database) dbest = database of expressed sequence tags dbsts = database of sequence tag sites gss = genomic survey sequences htgs = high throughput genomic sequence Step 4a: Select optional search parameters CD search Step 4a: Select optional search parameters Entrez! Filter Scoring matrix Word size Expect organism BLAST: 选择参数 ? 选择搜索的物种 ? 选择过滤条件，过滤掉查询序列中具有较低复杂度的掩盖部分 ? 选择记分矩阵。对于blastp有5种矩阵：PAM30，PAM70， BLOSUM45，BLOSUM62 (默认值)以及BLOSUM80。 ? 期望值的默认设定值是10。在这个E值下，随机出现得分等于或高于比对得分S的期望数为10个。当将期望选项值调小时，返回的数据库搜索结果将变少；匹配被搜索到的概率也会变小。增大E值将返回更多的结果? 字段长度。 默认值 BLASTp为3，BLASTn为11 ? 返回结果的格式 filtering Step 4b: optional formatting parameters Alignment view Descriptions Alignments taxonomy database query program taxonomy High scores low e values Cut-off: .05? 10-10? We will get to the bottom of a BLAST search in a few minutes… threshold score = 11 EVD parameters BLOSUM matrix Effective search space = mn = length of query x db length 10.0 is the E value gap penalties cut-off parameters General concepts How to evaluate the significance of your results How to handle too many results How to handle too few results BLAST search strategies Sometimes a real match has an E value 1 …try a reciprocal BLAST to confirm Sometimes a similar E value occurs for a short exact match and long less exact match Assessing whether proteins are homologous RBP4 and PAEP: Low bit score, E value 0.49, 24% identity (“twilight zone”). But they