Read Alignment

The analyse operations on an open set of reads: screen for leftover vector, align two reads head to head, open all of them as a multiple alignment, and phase a heterozygote into its two alleles.

What it doesCopy Anchor Link

Once your reads are trimmed and their base calls are as good as you can make them, the questions change from "is this read any good" to "what do these reads say". This section holds the operations that answer that, and all of them apply the edits and trims you have made rather than the raw file.

Screen for vector. Checks the read against a vector, adapter and primer database and reports what of it is not your insert. This is the commonest single-read question there is — is what I sequenced my clone or the backbone it came in? — so it is available with only one read open, unlike the alignment operations below.

Align 2 tracks. A head-to-head pairwise alignment of two open reads, reported as percent identity and a mismatch count, with the aligned bases displayed. Use it for a forward and reverse read of the same clone, or the same amplicon from two colonies.

Align all as MSA. Opens every read in the tab as a multiple sequence alignment. This is the path for three or more reads; the pairwise view stays available beside it because it is trace-aware and shows a head-to-head an MSA cannot.

Phase alleles. For a heterozygous read, splits the mixed base calls into the allele matching your open reference and the allele that does not, and reports the length of each.

When you'd use thisCopy Anchor Link

• After sequencing a clone: screen for vector first, then align the forward and reverse reads to each other, then compare against the designed construct in the verification tool.
• When a read is shorter than you expected — the vector screen usually explains it.
• When you have sequenced several colonies from one transformation: open them all in one tab and align them as an MSA to see which are the same and which are not.
• When a chromatogram shows double peaks at a position and you want the two underlying sequences rather than a string of ambiguity codes.

What this computesCopy Anchor Link

• A pairwise alignment of two reads by global Needleman–Wunsch with free end gaps — the right choice for two Sanger reads, which cover overlapping but not identical stretches of the same molecule, so penalising the overhang would push the aligner into inventing internal gaps to avoid it
• Percent identity over columns where BOTH reads have a base, gap columns excluded from numerator and denominator alike, so the number answers how often do they agree where they both have something to say rather than being diluted by overhang
• A mismatch count and the two aligned strings, drawn as an alignment track
• A vector screen over the reads, reporting matching regions
• Allele phasing: the primary and secondary base calls at every position are compared against the reference and each mixed position's two possibilities assigned to the reference and non-reference allele, so a heterozygous read becomes two sequences with a length each, instead of one string of ambiguity codes. It needs the secondary base calls that only an ABIF file carries
• A hand-off to MSA, opening every read as an alignment in its own canvas

Where it livesCopy Anchor Link

The Analyze section of the Chromatogram panel, acting on the active track and on the reads open in that tab. Its header carries a detach control that floats the whole section.

The alignment operations appear only when the tab has two or more reads, because they have nothing to work on otherwise. The vector screen sits outside that gate on purpose: it needs one read, and one read is the commonest case.

Inputs and outputsCopy Anchor Link

In: the reads open in the active trace tab, with their edits and trims applied; for phasing, the sequence open in the editor as the reference.

Out:

• The vector screen's report of matching regions.
• For the pairwise alignment: percent identity, a mismatch count and the two aligned strings, shown as an alignment track.
• For the MSA hand-off: all reads opened as an alignment in its own canvas.
• For phasing: the reference allele and the variant allele, with the length of each.

What it refuses, and what it says:

• Phasing with no reference open — you are told to open a reference sequence in the editor, since phasing is defined relative to one.
• Phasing a read it cannot phase — the method needs an ABIF (.ab1) read carrying user-edited base calls, and says exactly that rather than returning an empty result.
• Aligning with fewer than two reads — the controls are not shown, so there is no dead button.

ControlsCopy Anchor Link

The scienceCopy Anchor Link

The pairwise alignment is a global Needleman–Wunsch with free end gaps, which is the right choice for two Sanger reads: they cover overlapping but not identical stretches of the same molecule, so penalising the overhanging ends would push the aligner into inventing internal gaps to avoid them. Identity is computed over columns where both reads have a base — gap columns are excluded from both the numerator and the denominator — so the percentage is "how often do they agree where they both have something to say", not a figure diluted by overhang.

Allele phasing compares the primary and secondary base calls at every position against the reference and assigns each mixed position's two possibilities to the reference and the non-reference allele, so a heterozygous read becomes two sequences instead of one string of ambiguity codes. It requires the secondary base calls that only an ABIF file carries.

The algorithms are ports of the established Sanger analysis packages — the alignment and trace handling from SeqTrace and cfutils, the phasing from sangerseqR — and are checked against those originals byte for byte, so a phased allele or an identity figure here matches what those tools produce for the same file.

LimitationsCopy Anchor Link

• Two reads at a time in the pairwise view. A third read is the MSA path, which is a different view with different strengths — it is not trace-aware.
• Phasing needs an ABIF with edited base calls. A read from another format, or one whose secondary calls were never made, cannot be phased, and no approximation is offered in its place.
• Phasing is per position, not per haplotype. It assigns each mixed site to the reference or non-reference allele; it does not resolve which distant sites travel together on the same molecule, which no single Sanger read can.
• Percent identity is not a verdict. Two reads at 99% identity over 600 bases differ at six positions, and where those six sit matters more than the percentage.

RelatedCopy Anchor Link

In this group: Edit annotation · Enzyme · Insert sequence · Merge features · Manage enzymes · Dot plot · Dot plot grid · Melt curve · Amplification curves · Population genetics · RNA-seq · Site avoidance heat map · Protein coverage · Reference-gene stability · Digital PCR · Codon usage matrix · Pairwise distance distribution · Nearest-neighbor distances · GC content distribution · Base composition per cycle · Pairwise distance grid · Per-cycle channel occupancy · Grammar assembly plan · Gibson assembly plan · Assembly plan build order · Golden Gate assembly plan · Guide track · Primer binding track · Hydropathy profile · Charge vs pH · Amino-acid composition · Hydrophobic moment · Optimization profiles · Digest gel · Virtual gel · Initiation-window fold · Antibody domain map · Electropherogram · Circuit netlist · Circuit transfer functions · Circuit construct · Construction graph · Protocol · Read Quality Profile · Raw Signal · Verify vs Reference · Chromosomal microarray · Variant calls · Small molecule