Parsing sequence alignments into Hi-C pairs¶
Hi-C experiments aim to measure the frequencies of contacts between all pairs of loci in the genome. In these experiments, the spacial structure of chromosomes is first fixed with formaldehyde crosslinks, after which DNA is partially digested with restriction enzymes and then re-ligated back. Then, DNA is shredded into smaller pieces, released from the nucleus, sequenced and aligned to the reference genome. The resulting sequence alignments reveal if DNA molecules were formed through ligations between DNA from different locations in the genome. These ligation events imply that ligated loci were close to each other when the ligation enzyme was active, i.e. they formed “a contact”.
pairtools parse detects ligation events in the aligned sequences of
DNA molecules formed in Hi-C experiments and reports them in the .pairs/.pairsam
Throughout this document we will be using the same visual language to describe how DNA sequences (in the .fastq format) are transformed into sequence alignments (.sam/.bam) and into ligation events (.pairs).
Short-read sequencing determines the sequences of the both ends (or, sides) of DNA molecules (typically 50-300 bp), producing read pairs in .fastq format (shown in the first row on the figure above). In such reads, base pairs are reported from the tips inwards, which is also defined as the 5’->3’ direction (in accordance of the 5’->3’ direction of the DNA strand that sequence of the corresponding side of the read).
Alignment software maps both reads of a pair to the reference genome, producing alignments, i.e. segments of the reference genome with matching sequences. Typically, if the read length is not very large (< 150 bp), there will be only two alignments per read pair, one on each side. But, sometimes, the parts of one or both sides may map to different locations on the genome, producing more than two alignments per DNA molecule (see Multiple ligations (walks)).
pairtools parse converts alignments into ligation events (aka
Hi-C pairs aka pairs). In the simplest case, when each side has only one
unique alignment (i.e. the whole side maps to a single unique segment of the
genome), for each side, we report the chromosome, the genomic position of the
outer-most (5’) aligned base pair and the strand of the reference genome that
the read aligns to.
pairtools parse assigns to such pairs the type
Sometimes, one side or both sides of a read pair may not align to the reference genome:
In this case,
pairtools parse fills in the chromosome of the corresponding
side of Hi-C pair with
!, the position with
0 and the strand with
Such pairs are reported as type
NU (null-unique, when the other side has
a unique alignment) or
NN (null-null, when both sides lack any alignment).
Similarly, when one or both sides map to many genome locations equally well (i.e.
have non-unique, or, multi-mapping alignments),
pairtools parse reports
the corresponding sides as (chromosome=
MU (multi-unique) or
MM (multi-multi) or
depending on the type of the alignment on the other side.
pairtools parse calls an alignment to be multi-mapping when its
(which depends on the scoring gap between the two best candidate alignments for a segment)
is equal or greater than the value specied with the
--min-mapq flag (by default, 1).
Multiple ligations (walks)¶
If the read is long enough (e.g. larger than 150 bp), it may contain more than two alignments:
Molecules like these typically form via multiple ligation events and we call them
walks . The mode of walks reporting is controlled by
--walks-policy parameter of
A pair of sequential alignments on a single read is ligation junction. Ligation junctions are the Hi-C contacts
that have been directly observed in the experiment. They are reported in lower-case letters if walks policy
is set to
all (default). For details, wee Rescuing complex ligations
However, traditional Hi-C pairs do not have direct evidence of ligation
because they arise from read pairs that do not necessarily contain ligation junction.
To filter out the molecules with complex walks,
--walks-policy can be set to:
maskto tag these molecules as type
WW(single ligations are rescued, see Rescuing single ligations) ,
5anyto report the 5’-most alignment on each side,
5uniqueto report the 5’-most unique alignment on each side,
3anyto report the 3’-most alignment on each side,
3uniqueto report the 3’-most unique alignment on each side.
Rescuing complex ligations¶
The complex walks are DNA molecules containing more than one ligation junction that may end up in more than one alignment on forward, reverse, or both reads:
pairtools parse detects such molecules and rescues them with
Briefly, the algorithm of complex ligation walks rescue detects all the unique ligation junctions, and do not report
the same junction as a pair multiple times. Importantly, these duplicated pairs might arise when both forward and reverse
reads read through the same ligation junction. However, these cases are successfully merged by
To restore the sequence of ligation events, there is a special field
junction_index that can be reported as
a separate column of .pair file by setting
--add-junction-index. This field contains information on:
- the order of the junction in the recovered walk, starting from 5’-end of forward read
- type of the junction:
- “u” - unconfirmed junction, right and left alignments in the pair originate from different reads (forward or reverse). This might be indirect ligation (mediated by other DNA fragments).
- “f” - pair originates from the forward read. This is direct ligation.
- “r” - pair originated from the reverse read. Direct ligation.
- “b” - pair was sequenced at both forward and reverse read. Direct ligation.
With this information, the whole sequence of ligation events can be restored from the .pair file.
Rescuing single ligations¶
Importantly, some of DNA molecules containing only one ligation junction may still end up with three alignments:
A molecule formed via a single ligation gets three alignments when one of the two ligated DNA pieces is shorter than the read length, such that that read on the corresponding side sequences through the ligation junction and into the other piece . The amount of such molecules depends on the type of the restriction enzyme, the typical size of DNA molecules in the Hi-C library and the read length, and sometimes can be considerable.
pairtools parse detects such molecules and rescues them (i.e.
changes their type from a walk to a single-ligation molecule). It tests
walks with three aligments using three criteria:
- On the side with two alignments (the chimeric side), the “inner” (or, 3’) alignment must be on the same chromosome as the alignment on the non-chimeric side.
- The “inner” alignment on the chimeric side and the alignment on the non-chimeric side must point toward each other.
- These two alignments must be within the distance specified with the
--max-molecule-sizeflag (by default, 2000bp).
Sometimes, the “inner” alignment on the chimeric side can be non-unique or “null”
(i.e. when the unmapped segment is longer than
as described in Interpreting gaps between alignments).
pairtools parse ignores such alignments
altogether and thus rescues such walks as well.
Interpreting gaps between alignments¶
Reads that are only partially aligned to the genome can be interpreted in two different ways. One possibility is to assume that this molecule was formed via at least two ligations (i.e. it’s a walk) but the non-aligned part (a gap) was missing from the reference genome for one reason or another. Another possibility is to simply ignore this gap (for example, because it could be an insertion or a technical artifact), thus assuming that our molecule was formed via a single ligation and has to be reported:
Both options have their merits, depending on a dataset, quality of the reference
genome and sequencing.
pairtools parse ignores shorter gaps and keeps
longer ones as “null” alignments. The maximal size of ignored gaps is set by
--max-inter-align-gap flag (by default, 20bp).
|||Following the lead of C-walks|
|||This procedure was first introduced in HiC-Pro and the in Juicer .|