Date post: | 19-Jan-2016 |
Category: |
Documents |
Upload: | taylor-albert |
View: | 224 times |
Download: | 0 times |
S C H O O L O F C O M P U T I N GA N D R E W M A X W E L L
9 / 1 1 / 2 0 1 3
SEQUENCE ALIGNMENT AND COMPARISON BETWEEN BLAST AND
BWA-MEM
OUTLINE
• BLAST• BWA-MEM• Comparisons
BLAST
• Basic Local Alignment Search Tool• Developed by NCBI• NCBI - National Center for Biotechnology Information• NLM – US National Library of Medicine• NIH – National Institute of Health• http://blast.ncbi.nlm.nih.gov/
• Latest Version (executable)• 2.2.28+• ftp://ftp.ncbi.nlm.nih.gov/blast+/LATEST/
BLAST
• A suite of tools that work together to search for similar sequences of different protein or nucleotide DNA sequences.• Three Categories of Applications
1. Search Tools2. BLAST Database Tools3. Sequence Filtering Tools
• BLAST Command Line User Manual• http://www.ncbi.nlm.nih.gov/books/NBK1763/
SEARCH APPLICATIONS
• Execute a BLAST search.• blastn – Nucleotide Blast• Nucleotide database using nucleotide query.
• blastp - Protein Blast• Protein database using protein query.
• blastx • Protein database using translated nucleotide query.
• tblastx• Translated nucleotide database using a translated
nucleotide query.
• tblastn• Translated nucleotide database using a protein query.
SEARCH APPLICATIONS CONT.
• psiblast• Position-Specific Iterated BLAST• Finds sequences significantly similar to the query in a
database search and uses the resulting alignments to build a Position-Specific Score Matrix (PSSM).
• rpsblast• Reverse Position-Specific BLAST• Uses a query to search a database of pre-calculated
PSSMs and report significant hits in a single pass.
• rpstblastn• Searches database using a translated nucleotide query.
BLAST DATABASE APPLICATIONS
• Create or examine BLAST databases.• makeblastdb• Creates BLAST databases.
• blastdb_aliastool• Manage BLAST databases.• Search multiple databases together or search a subset of
sequences within a database.
• makeprofiledb• Builds an RPS-BLAST database.
• blastdbcmd• Examine the contents of a BLAST database.
SEQUENCE FILTERING APPLICATIONS
• Segmasker• Identifies and masks low complexity regions* of protein sequences.
• Dustmasker• Similar to segmasker but for nucleotide sequences.
• Windowmasker• Uses a genome to identify sequences represented too often to be of
interest to most users.
• *Low-Complexity Regions – Regions of a sequence composed of few elements.• These will be ignored by BLAST unless explicitly told to include them
in searches.• May achieve high scores that may bump more significant sequences.
BLAST ALGORITHM
http://www.ncbi.nlm.nih.gov/books/NBK62051/bin/blastpic1.jpg
E-VALUE
• The number of hits to see by chance when searching the database.• This value decreases exponentially when the
score is increased.• The lower the e-value is, the more significant the
match is.• This also depends on the length of the query
sequence. E-values will be higher with shorter sequences because there is a higher probability of a query sequence occurring in the database by chance.
BITSCORE
• The bitscore value is derived from the raw alignment score S.
• Lambda and K are statistical parameters of the scoring system.
http://www.ncbi.nlm.nih.gov/books/NBK21106/bin/glossfig1.jpg
EXAMPLE RUN
FASTA FORMAT
• Text-based format representing nucleotide or peptide sequences.• A “>”, followed by the sequence identifier, then
an optional description.
• >seq_1 Some description• GAGGGCTCATCCGGGAATCGAACCCGGGACCTCTCG
CACCCTAAGCGAGAATCATACGACTAGACCAATGAGCCGTGTTCAAAGAGTGTCAAAATGTGTTTCGAGCGTCTATGTCCAAAGTGAATTGCTTGTCTTTTGAGTTTTGCGATTG
SAMPLE OUTPUT
BWA-MEM
• Burrows-Wheeler Aligner• A software package for aligning sequences
against large reference genomes.• The BWA package contains three different
algorithms: BWA-backtrack, BWA-SW, and BWA-MEM.• Manual Page• http://bio-bwa.sourceforge.net/bwa.shtml
BWA-MEM
• Can align 70bp to 1Mbp• MEM – Maximal Exact Matches• Local alignment
HOW TO RUN
• Index the reference FASTA file.• Run BWA-MEM with a query file (in FASTQ format)
against the reference database.• The output is in a SAM file format.
FASTQ FORMAT
• Similar to a FASTA format, but with a quality score added.• @HWI-EAS397:8:1:1067:18713#CTTGTA/1• TGGAGATGAGATTGTCGGCTTTATTACCCAGGGGCG
GGGGGTTATTGTA• +• Y^]Lcda]YcffccffadafdWKd_V\``^\
aa^BBBBBBBBBBBBBBB• The quality score is an integer mapping of the
probability that the base is incorrect.
SAM FILE
• Eleven mandatory fields and a variable amount of optional fields.• The optional fields are a key-value pair of
TAG:TYPE:VALUE. These store extra information.
SAM REQUIRED FIELDS
SAM OPTIONAL FIELDS
BWA-MEM ALGORITHM
• Seeds alignments with maximal exact matches• Then, uses affine-gap Smith-Waterman algorithm.
http://en.wikipedia.org/wiki/Smith%E2%80%93Waterman_algorithm
BWA-MEM OPTIONS
• t – Number of threads• T – Don’t output alignment with score lower than
INT.• a – Output all found alignments for single-end or
unpaired paired-end reads.
• (In output, ‘*’ are considered zero.)
EXAMPLE RUN
SAMPLE OUTPUT
REFERENCES
• NCBI Help Manual - http://www.ncbi.nlm.nih.gov/books/NBK3831/• Bwa - http://bio-bwa.sourceforge.net/• FASTA - http://en.wikipedia.org/wiki/FASTA_format• FASTQ - http://en.wikipedia.org/wiki/FASTQ_format• Li, H, et al. (2009). The Sequence Alignment/Map
format and SAMtools. Vol. 25 no 16, Bioinformatics Applications Note.