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

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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.