Magic-BLAST is a tool for mapping large next-generation RNA or DNA sequencing
runs against a whole genome or transcriptome. Unlike other BLAST nucleotide search programs, such as BLASTN or Megablast, Magic-BLAST produces spliced alignments and optimizes alignment scores for paired reads. 

Magic-Blast incorporates within the NCBI BLAST code framework ideas
developed in the NCBI Magic pipeline, in particular hit extensions by
local walk and jump, which is faster than Smith-Waterman extension
(http://www.ncbi.nlm.nih.gov/pubmed/26109056).

We call the whole next generation run (from Illumina, Roche-454, ABI, or
another sequencing platform excluding SOLiD), a query. The input reads may
be provided as SRA accession or file in a SRA, FASTA, FASTQ, or FASTC format.
Read pairs can be presented as parallel files, or as successive reads in a
single file.

The reference genome or transcriptome can be given as a BLAST database
or a FASTA file. It is preferable to use BLAST database for large genomes,
such as human, or transcript collections, such as all of RefSeq, Ensembl,
or AceView. The procedure for creating a BLAST database is described below.

The full list of options is listed when you use -help option.

For more information, visit https://ncbi.github.io/magicblast/


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EXAMPLES:
Use this command line to map RNA-seq reads in FASTA format to a reference
genome in FASTA format:
magicblast -query reads.fa -subject genome.fa

Use this command line to map RNA-seq reads to a reference genome provided
as a BLAST database:
magicblast -query reads.fa -db genome

Use this command line to map RNA-seq reads in FASTQ format:
magicblast -query reads.fastq -db genome -infmt fastq

The reads can also be provided from the standard input:
cat reads.fa | magicblast -query - -db genome
cat reads.fa | magicblast -db genome

Magic-Blast recognizes read and reference file names with extension ".gz"
as compressed and decompresses them automatically using gzip.

Use this command line to map SRA run to a genome:
magicblast -sra sra_accession -db genome



PAIRED-READS:

For paired reads presented as successive entries in a single FASTA or FASTQ
file, i.e. read 1 and 2 of fragment 1, then read 1 and 2 of fragment 2,
etc., simply add the parameter '-paired':
magicblast -query reads.fa -db genome -paired

If you are using fastq-dump
(http://www.ncbi.nlm.nih.gov/Traces/sra/sra.cgi?view=toolkit_doc&f=fastq-dump)
to download NCBI SRA data or convert SRA file to the FASTQ format, use these
parameters for runs with paired reads to create a single merged input file
for Magic-Blast:
fastq-dump -I --split-spot <accession or file>

For paired reads presented in two parallel files, use these options:
magicblast -query reads.fa -query_mate mates.fa -db genome


RNA versus DNA:

By default, Magic-Blast aligns RNA reads to a genome and reports spliced
alignments, possibly spanning several exons. To disable spliced alignments,
use the '-splice F' option. Use the '-reftype transcriptome' option, to
map reads to a transcriptome database. These are example command lines:
magicblast -query reads.fa -db genome -splice F
magicblast -query reads.fa -db genome -reftype transcriptome

Magic-Blast finds alignments between a read and a genome based on initial
common word in both. To make mapping faster we first count word occurrences
in the genome and disregard those that occur too often. With the
'-reftype transcripts' option the words that are frequent in the transcript
database are used for alignment.


MULTI-THREADING

To use multiple CPUs, specify the maximal number of threads with the
'-num_threads' parameter:
magicblast -query reads.fa -db genome -num_threads 10


OUTPUT:

By default, results are provided to the standard output in the SAM format.
Use '-out filename' option to redirect output to a file.
Use '-outfmt' option to specify the output format:
-outfmt SAM : SAM format (default)
-outfmt tabular : exports a simple tab delimited format defined below.

The output can be also compressed, using the '-gzo' flag:
magicblast -query reads.fa -db genome -out output.gz -gzo


CREATION OF A BLAST DATABASE:

Use this command line to create a BLAST database:
makeblastdb -in fasta_file -dbtype nucl -parse_seqids -out database_name
-title "Database title"

The -parse_seqids option is required to keep the original sequence
identifiers. Otherwise makeblastdb will generate its own identifiers.


TABULAR OUTPUT FORMAT

The tabular output format shows one alignment per line with these tab
delimited fields:
1. Query/read sequence identifier
2. Reference sequence identifier
3. Percent identity of the alignment
4. Not used
5. Not used
6. Not used
7. Alignment start position on the query sequence
8. Alignment stop position on the query sequence
9. Alignment start position on the reference sequence
10. Alignment stop position on the reference sequence
11. Not used
12. Not used
13. Alignment score
14. Query strand
15. Reference sequence strand
16. Query/read length
17. Alignment as extended BTOP string
    This is the same BTOP string as in BLAST tabular output with a
    few extensions:
    - a number represents this many matches,
    - two bases represent a mismatch and show query and reference base,
    - base and gap or gap and base, show a gap in query or reference,
    - ^<number>^ represents an intron of this number of bases,
    - _<number>_ represents a gap in query of this number of bases,
    - (<number>) shows number of query bases that are shared between
    two parts of a spliced alignment; used when proper splice sites
    were not found
18. Number of different alignments reported for this query sequence
19. Information about detected splice sites, polyA tails, or adapter
    sequences found at the edges of the alignment
20. Compartment - a unique identifier for all alignments that belong to
    a single fragment. These can be two alignments for a pair of reads
    or alignments to exons that were not spliced.
21. Reverse complemented unaligned query sequence from the beginning
    of the query, or '-' if the query aligns to the left edge
22. Unaligned sequence at the end of the query, or '-'
23. Reference sequence identifier where the mate is aligned, if
    different from the identifier in column 2, otherwise '-'
24. Alignment start position on the reference sequence for the mate, or
    '-' if no alignment for the mate was found; a negative number
    denotes a divergent pair
25. Composite alignment score for all exons that belong to the fragment

Thank you for testing this code and providing us with feedback. Please,
let us know of any desired option, problem or difficulty.

E-mail blast-help@ncbi.nlm.nih.gov with questions or comments.
