# last-split

This program estimates "split alignments" (typically for DNA) or "spliced alignments" (typically for RNA).

It reads candidate alignments of query sequences to a genome, and looks for a unique best alignment for each part of each query. It allows different parts of one query to match different parts of the genome. This is useful for DNA queries that cross rearrangement breakpoints, or RNA queries that cross splice junctions.

## Examples

### Split alignment of DNA reads to a genome

Assume the DNA reads are in a file called "q.fastq" (in fastq-sanger format), and the genome is in "genome.fasta" (in fasta format). We can do the alignment like this:

    lastdb -uNEAR -R01 db genome.fasta
    lastal -Q1 db q.fastq | last-split > out.maf

### Spliced alignment of RNA reads to a genome

Now we assume that "q.fastq" has reads from RNA forward (sense) strands. This time, we provide the genome information to last-split, which causes it to do spliced instead of split alignment, and also tells it where the splice signals are (GT, AG, etc):

    lastdb -uNEAR -R01 db genome.fasta
    lastal -Q1 -D10 db q.fastq | last-split -g db > out.maf

This will favour splices starting at GT (and to a lesser extent GC and AT), and ending at AG (and to a lesser extent AC). However, it allows splices starting and ending anywhere. It also favours splices with introns of typical length, specified by a log-normal distribution (i.e. cis-splices). However, it allows arbitrary trans-splices between any two places in the genome.

-D10 sets a very loose significance threshold, so that we can find very short parts of a spliced alignment (e.g. short exons). Note that last-split discards the lowest-significance alignments, but it uses them to estimate the ambiguity of higher-significance alignments.

If your reads are from unknown/mixed RNA strands, add -d2 to the last-split options.

### Alignment of two whole genomes

We can align the cat and rat genomes like this:

    lastdb -uMAM8 -cR11 catdb cat.fasta
    lastal -m100 -E0.05 catdb rat.fasta | last-split -m1 > out.maf

This will align each rat base-pair to at most one cat base-pair, but not necessarily vice-versa. We can get 1-to-1 alignments by swapping the sequences and running last-split again:

    maf-swap out.maf | last-split -m1 > out2.maf

## FAQ

Q: | Before aligning RNA, should poly-A tails be trimmed?
A: | It's not essential, but it might make things faster. Poly-A tracts tend to have many matches in the genome. By trimming, you can prevent lastal and last-split from wasting time on such matches.

## Going faster by parallelization

For example, split alignment of DNA reads to a genome:

    parallel-fastq "lastal -Q1 db | last-split" < q.fastq > out.maf

This requires GNU parallel to be installed (http://www.gnu.org/software/parallel/).

## Output

The output is in MAF(-like) format:

    a score=150 mismap=0.000413
    s chr21  15963638 25 + 48129895 TCAGATGAGGACCTAATTTATTACT
    s query7       50 25 +       75 TCAGATGAGGACCTAATTTATTACT
    q query7                        EBEEC@CE=EEE?FEDAED5?@@D@
    p                               !#$'BBBBBBBBBBBBBBBBBBBBB

The "mismap" is the estimated probability that this part of the query should be aligned to a different part of the genome. The line starting with "p" indicates the probability that each base should be aligned to a different part of the genome. It uses a compact code:

> Symbol | Error probability | Symbol | Error probability
> `!` | 0.79 -- 1 | `0` | 0.025 -- 0.032
> `"` | 0.63 -- 0.79 | `1` | 0.02 -- 0.025
> `#` | 0.5 -- 0.63 | `2` | 0.016 -- 0.02
> `$` | 0.4 -- 0.5 | `3` | 0.013 -- 0.016
> `%` | 0.32 -- 0.4 | `4` | 0.01 -- 0.013
> `&` | 0.25 -- 0.32 | `5` | 0.0079 -- 0.01
> `'` | 0.2 -- 0.25 | `6` | 0.0063 -- 0.0079
> `(` | 0.16 -- 0.2 | `7` | 0.005 -- 0.0063
> `)` | 0.13 -- 0.16 | `8` | 0.004 -- 0.005
> `*` | 0.1 -- 0.13 | `9` | 0.0032 -- 0.004
> `+` | 0.079 -- 0.1 | `:` | 0.0025 -- 0.0032
> `,` | 0.063 -- 0.079 | `;` | 0.002 -- 0.0025
> `-` | 0.05 -- 0.063 | `<` | 0.0016 -- 0.002
> `.` | 0.04 -- 0.05 | `=` | 0.0013 -- 0.0016
> `/` | 0.032 -- 0.04 | `>` | 0.001 -- 0.0013

Other symbols indicate lower error probabilities, and "~" is the lowest possible. In general:

    Error probability <= 10 ^ -((ASCII value - 33) / 10)

The "mismap" is simply the lowest probability from the "p" line. (If you run last-split twice, as in the genome alignment example, the mismap is the lowest combined error probability from both "p" lines.)

## Split versus spliced alignment

Here is a split alignment:

    Query         ttctttgat--gctagtcctgatgttatggtattttttatcgaatgataa
                    |||||||--||||||                |||x||||||||||||
    Genome chrA  ...ctttgatatgctagt...             |||x||||||||||||
    Genome chrB                                 ...tttatatcgaatgata...

And here is a spliced alignment:

    Query        ctagtcgatatt--gctgtacgtctgttagctat-tttttcctctgtttg
                    |||x|||||--|||||||||----|||||||-|||||x|||||
    Genome chrA  ...gtctatattatgctgtacgt... |||||||-|||||x|||||
    Genome chrB                          ...tagctatattttttctctg...

Split alignment allows arbitrarily large unaligned parts in the middle of the query, whereas spliced alignment applies a standard gap penalty. (Both allow arbitrarily large unaligned parts at the edges of the query.)

## Specialized examples

### Faster spliced alignment

Spliced alignment can be slow. It can be sped up, at a small cost in accuracy, by not favouring cis-splices:

    lastdb -uNEAR -R01 db genome.fasta
    lastal -Q1 -D10 db q.fastq | last-split -c0 -t0.004 -g db > out.maf

The -c0 turns off cis-splicing, and the -t0.004 specifies a higher probability of trans-splicing.

### "Spliced" alignment of DNA reads to a genome

If we do not wish to allow arbitrarily large unaligned parts in the middle of the query, we can do "spliced" alignment without considering splice signals or favouring cis-splices:

    lastdb -uNEAR -R01 db genome.fasta
    lastal -Q1 db q.fastq | last-split -c0 > out.maf

## Options

> `-h, --help` | Show a help message, with default option values, and exit.
> `-f, --format=FMT` | Choose the output format: MAF+ (the default), or MAF (which omits the last-split "p" lines). The format name is not case-sensitive.
> `-g, --genome=NAME` | Do spliced alignment, and read splice signals (GT, AG, etc) from the named genome. NAME should be the name of a lastdb database.
> `-d, --direction=D` | Do spliced alignment, and set the strandedness of the queries: 0=antisense, 1=sense, 2=unknown/mixed. This determines whether forward and/or reverse-complement splice signals are used. If you use -d2, the output will have an extra "sense" field, indicating the log-odds that the query is sense-stranded:
>     log2[ prob(sense) / prob(antisense) ]
> `-c, --cis=PROB` | Do spliced alignment, and set the average probability per base of cis-splicing. The default value roughly fits human RNA.
> `-t, --trans=PROB` | Do spliced alignment, and set the average probability per base of trans-splicing.
> `-M, --mean=MEAN` | Do spliced alignment, and set the mean of ln(intron length). The default value fits human RNA.
> `-S, --sdev=SDEV` | Do spliced alignment, and set the standard deviation of ln(intron length). The default value fits human RNA.
> `-m, --mismap=PROB` | Don't write alignments with mismap probability > PROB. Low-confidence alignments will be discarded unless you increase this value!
> `-s, --score=INT` | Don't write alignments with score < INT. For SPLIT alignment, the default value is e (the lastal score threshold). Alignments with score just above INT will get high mismap probabilities. For SPLICED alignment, the default value is e + t * ln(100), where t is a scale factor that is written in the lastal header. This roughly means that, for every alignment it writes, it has considered alternative alignments with one-hundredth the probability. Alignments with score just above INT will not necessarily get high mismap probabilities.
> `-n, --no-split` | Do probability calculations as usual, but write the original alignments, annotated with "p" lines and mismap probabilities. Note that the mismap and score limits still apply.
> `-b, --bytes=B` | Skip any query sequence that would require more than B bytes of memory to process. (This only limits the size of some core data-structures: the total memory use will be greater.) A warning is written for each skipped sequence. You can use suffixes such as K (KibiBytes), M (MebiBytes), G (GibiBytes), T (TebiBytes), e.g. -b20G.
> `-v, --verbose` | Show progress information on the screen.
> `-V, --version` | Show version information and exit.

## Details

- The input must be in MAF format, and it must include header lines (of the kind produced by lastal) describing the alignment score parameters.

- The program reads one batch of alignments at a time (by looking for lines starting with "# batch"). If the batches are huge (e.g. because there are no lines starting with "# batch"), it might need too much memory.

- lastal can optionally write "p" lines, indicating the probability that each base is misaligned due to wrong gap placement. last-split, on the other hand, writes "p" lines indicating the probability that each base is aligned to the wrong genomic locus. You can combine both sources of error (roughly) by taking the maximum of the two error probabilities for each base.

The following points matter only if you are doing something unusual (e.g. bisulfite alignment):

- If the header has more than one score matrix, last-split will use the first one.

- It assumes this score matrix applies to all alignments, when the alignments are oriented to use the forward strand of the query.

## last-split8

last-split8 is almost identical to last-split. The only difference is the -g option: last-split can only read the output of lastdb, whereas last-split8 can only read the output of lastdb8.

## Limitations

last-split does not support:

- Generalized affine gap costs.

## To do

- An option to specify splice signals and their strengths.