Numerous sequencing assays encode interesting biology in various properties of read alignments withu nucleotide precision. For example:

• ribosome profiling encodes the positions of ribosomal P-sites as a function of both read length and alignment coordinates ([IGNW09][ILW11])
• Bisulfite sequencing encodes methylation status as C-to-T transitions within read alignments
• DMS-Seq ([RZW+14]) and Pseudouridine profiling (:cite:) encode unstructured regions of RNA or sites of pseudouridine modification, respectively, in the 5’ termini fo their read aligments.

plastid uses configurable mapping functions to decode the biology of interest from read alignments, and convert the decoded data into arrays over regions of interest (such as an array of the number of ribosomes at each nucleotide in a coding region).

This design enables plastid‘s tools to operate on sequencing data from virtually any NGS assay, provided the appropriate mapping function and parameters.

Mapping functions are described in the following sections:

## Mapping functions included in plastid¶

At present, the following mapping functions are provided, although users are encouraged to write their own mapping functions as needed. These include:

Fiveprime end mapping
Each read alignment is mapped to its 5’ end, or at a fixed offset (in nucleotides) from its 5’ end
Variable fiveprime end mapping

Each read alignment is mapped at a fixed distance from its 5’ end, where the distance is determined by the length of the read alignment.

This is used for ribosome profiling of yeast ([IGNW09]) and mammalian cells ([ILW11]).

Stratified variable fiveprime end mapping

This multidimensional mapping function behaves as variable fiveprime end mapping, and additionally returns arrays that are stratified by read length.

For each region of interest, a 2D array is returned, in which each row represents a given read length, and each column a nucleotide position in the region of interest. In other words, summing over the columns produces the same array that would be given by variable fiveprime end mapping.

Threeprime end mapping
Each read alignment is mapped to its 3’ end, or at a fixed offset (in nucleotides) from its 3’ end.
Entire or Center-weighted mapping

Zero or more positions are trimmed from each end of the read alignment, and the remaining N positions in the alignment are incremented by 1/N read counts (so that each read is still counted once, when integrated over its mapped length).

This is also used for ribosome profiling of E. coli ([OBS+11]) and D. melanogaster ([DFB+13]), and RNA-seq.

In the image below, the same set of read alignments from a ribosome profiling experiment is mapped under various functions. Note the start codon peak and stop codon peak that appear when reads are mapped to specific locations:

Top: gene model. Middle: alignments of ribosome-protected footprints, displayed as in the IGV genome browser without a mapping function. Bottom rows: Ribosome footprints mapped under various mapping functions.

### Choosing mapping functions in command-line scripts¶

Mapping functions may be specified to command-line scripts using the following command-line arguments (note, command-line scripts don’t at present support mapping functions that return multidimensional arrays):

 Mapping function Argument Fiveprime --fiveprime Fiveprime variable --fiveprime_variable Threeprime --threeprime Center/entire --center

The following arguments additionally influence how mapping functions behave:

 Argument Behavior --offset X For --fiveprime or --threeprime, X is taken to be an integer specifying the offset into the read, at which read alignments should be mapped. For --fiveprime_variable, X is taken to be the filename of a two-column tab-delimited text file, in which first column represents read length or the special keyword ‘default’, and the second column represents the offset from the five prime end at which reads of that length should be mapped. --nibble X X is taken to be the number of bases to trim from each end of the read before mapping.

See the documentation for individual command-line scripts for a detailed discussion of their arguments.

### Choosing mapping functions in interactive Python sessions¶

Mapping functions in plastid are applied when read alignments are imported. Read alignments are held in data structures called GenomeArrays (see plastid.genomics.genome_array).

Alignments in BAM format can be imported into a BAMGenomeArray. Mapping functions are set via set_mapping():

>>> from plastid.genomics.genome_array import BAMGenomeArray, FivePrimeMapFactory, CenterMapFactory

>>> alignments = BAMGenomeArray("SRR609197_riboprofile_5hr_rep1.bam")

>>> # map reads 5 nucleotides downstream from their 5' ends
>>> alignments.set_mapping(FivePrimeMapFactory(offset=5))


and, the mapping function for a BAMGenomeArray can be changed at any time:

>>> # map reads along entire lengths
>>> alignments.set_mapping(CenterMapFactory())


Alignments in bowtie format can be imported into a GenomeArray. Because bowtie files are not sorted or indexed, mapping functions must be applied upon import, and cannot be changed afterwards:

>>> from plastid.genomics.genome_array import GenomeArray, five_prime_map

>>> # map reads 5 nucleotides downstream from their 5' ends
>>> fiveprime_alignments = GenomeArray()

>>> # map reads along entire lengths
>>> entire_alignments = GenomeArray()


Method names for the various mapping functions appear below:

 Mapping function GenomeArray, SparseGenomeArray BAMGenomeArray Fiveprime five_prime_map() FivePrimeMapFactory Fiveprime variable variable_five_prime_map() VariableFivePrimeMapFactory Stratified fiveprime variable not implemented StratifiedVariableFivePrimeMapFactory Threeprime three_prime_map() ThreePrimeMapFactory Center/entire center_map() CenterMapFactory

## Writing new mapping functions¶

Mapping functions in plastid are implemented as callables (functions or callable class instances). Mapping functions for BAMGenomeArray require the following signatures:

### Parameters¶

alignments
A list of read alignments represented as pysam.AlignedSegment objects. These correspond to the alignments that will be mapped. Typically, these overlap segment.
segment
A GenomicSegment corresponding to a region of interest

### Return values¶

list
A list of read alignments (pysam.AlignedSegment) that map within segment under the mapping function implemented by the function.
numpy.ndarray

An array of values at each position in segment, from left-to-right / lowest-to-highest coordinates relative to the genome (not relative to the segment).

More generally, if the mapping function returns a multi-dimensional array, the last dimension must represent the positions in segment (e.g., for a 2D array, the columns would represent the nucleotide positions).

### Example 1: Fiveprime alignment mapping¶

This mapping function maps read alignments to their 5’ ends, allowing an optional offset:

>>> import numpy
>>> import warnings

>>> def fiveprime_map_function(alignments,segment,offset=0):
>>>     count_array = numpy.zeros(len(segment))
>>>                           UserWarning)
>>>
>>>         # count offset 5' to 3' if the segment is on the plus-strand
>>>         # or is unstranded
>>>         if segment.strand == in ("+","."):
>>>         # count offset from other end if segment is on the minus-strand
>>>         else:
>>>             p_site = read.positions[-offset - 1]
>>>
>>>         if p_site >= segment.start and p_site < segment.end:
>>>             count_array[p_site - seg.start] += 1
>>>


But, BAMGenomeArray will only pass the parameters alignments and segment to mapping functions. To specify an offset, use a wrapper function:

>>> def MyFivePrimeMapFactory(offset=0):
>>>    def new_func(alignments,segment):
>>>       return fiveprime_map_function(alignments,segment,offset=offset)
>>>
>>>    return new_func

>>> alignments = BAMGenomeArray("SRR609197_riboprofile_5hr_rep1.bam")
>>> alignments.set_mapping(MyFivePrimeMapFactory(offset=5))


### Example 2: mapping alignments to their mismatches¶

BAM files contain rich information about read alignments, and these are exposed to us via pysam.AlignedSegment. This mapping function maps read alignments to sites where they mismatch a reference genome, assuming the alignment contains no indels. Mismatch information is pulled from the MD tag for each read alignment:

>>> import re
>>> nucleotides = re.compile(r"[ACTGN]")
>>>
>>> def mismatch_mapping_function(alignments,segment):
>>>     count_array = numpy.zeros(len(segment))
>>>             # we are also assuming no indels, which would make parsing MD more complicated.
>>>             #
>>>             # mismatches are in stored in MD tag of reach alignment in SAM/BAM files
>>>             # for see MD tag structure http://samtools.sourceforge.net/SAM1.pdf
>>>             # they basically look like numbers of matches separated by
>>>             # the letter that mismatches. e.g. 12A15C22
>>>             # means: 12 matches, followed by mismatch 'A', followed by 15 matches,
>>>             #        followed by mismatch 'C', followed by 22 matches
>>>             #
>>>             # convert MD tag to a vector of positions that mismatch
>>>             if tag == "MD":
>>>                 mismatched_positions  = numpy.array([int(X) for X in re.split(nucleotides,val)[:-1]])
>>>                 mismatched_positions += numpy.arange(len(mismatched_positions))
>>>
>>>         # figure out coordinate of mismatch with respect to genome and segment
>>>         for pos in mismatched_positions:

>>> alignments.set_mapping(mismatch_mapping_function)