DNA Aligning

The global option controls whether alignment is forced to be end-to-end in the read. The available values are 0 or 1.

For long reads, the value 0 is preferred, so significant read segments after a break can be clipped without severely decreasing the alignment score. Examples of breaks include a large indel, structural variant, chimeric read, and so forth. Setting the option to 1 might not have the desired effect with longer reads, because insertions at or near the ends of a read can function as pseudoclipping. Also, when global is 0, multiple (chimeric) alignments can be reported when various portions of a read match widely separated reference positions.

For short reads, setting global to 1 is sometimes preferable. Short reads are unlikely to overlap structural breaks, unable to support chimeric alignments, and are suspected of incorrect mapping if they cannot align well end-to-end.

To make a soft preference for unclipped alignments, consider using the unclip-score option, or increasing it, instead of setting global to 1.

The match-score option specifies the score for a read nucleotide matching a reference nucleotide (A, C, G, or T), or matching a reference 2–3 nucleotide IUPAC-IUB code. The value is an unsigned integer from 0 to 15. Only set the match_score option to 0 when global is 1. A higher match score results in longer alignments and fewer long insertions.

The match-n-score option specifies the score for an aligned position where the read position and/or the reference position is an N code. The option is a signed integer from -16 to 15.

The mismatch-pen option sets the penalty, or negative score, for a read nucleotide mismatching any reference nucleotide or IUPAC-IUB code, except N. The option is an unsigned integer from 0 to 63. A higher mismatch penalty results in alignments with more insertions, deletions, and clipping to avoid SNPs.

The gap-open-pen option set the penalty, or negative score, for opening a gap (ie, an insertion or deletion). The value is only for a 0-base gap. The penalty is always added to the gap length multiplied by gap-ext-pen. The option is an unsigned integer from 0 to 127. A higher gap open penalty causes fewer insertions and deletions of any length in alignment CIGARs. Clipping or alignment through SNPs is used instead.

The gap-ext-pen option sets the penalty, or negative score, for extending a gap (ie, an insertion or deletion) by one base. This option is an unsigned integer from 0 to 15. A higher gap extension penalty causes fewer long insertions and deletions in alignment CIGARs. Short indels, clipping, or alignment through SNPs is used instead.

The unclip-score option sets the score bonus for an alignment reaching the beginning or end of the read. A higher unclipped bonus causes alignment more often to reach the beginning and/or end of a read, where alignment can be done without too many SNPs or indels. An end-to-end alignment receives twice the bonus. The option is an unsigned integer from 0 to 127.

A nonzero unclip-score is useful when global is 0 to make a soft preference for unclipped alignments. Unclipped bonuses have little effect on alignments when global is 1, because end-to-end alignments are forced. However, 2 × unclip-score does add to every alignment score unless no-unclip-score is 1).

For longer reads, setting unclip-score much higher than gap-open-pen can result in insertions at or near one end of a read being used as pseudoclipping, as happens with global is 1.

The no-unclip-score option can be 0 or 1. The default is 1. When no-unclip-score is set to 1, any unclipped bonus (unclip-score) contributing to an alignment is removed from the alignment score before further processing. Unclipped bonuses can include comparison with aln-min-score, comparison with other alignment scores, and reporting in AS or XS tags. However, the unclipped bonus still affects the best-scoring alignment found by the Smith-Waterman alignment to a given reference segment, biasing toward unclipped alignments.

If a unclip-score greater than 0 causes a Smith-Waterman local alignment to extend out to one or both ends of the read, the following score changes are possible:

The default, no-unclip-score is 1, is recommended when global is 1, because every alignment is end-to-end. There is no need to add the same bonus to every alignment.

When changing no-unclip-score, consider whether aln-min-score should be adjusted. When no-unclip-score is 0, unclipped bonuses are included in alignment scores compared to the aln-min-score floor, so the subset of alignments filtered out by aln-min-score can change significantly with no-unclip-score.

The aln-min-score option specifies a minimum acceptable alignment score. Any alignment results scoring lower are discarded. Increasing or decreasing aln-min-score can reduce or increase the percentage of reads mapped. This option is a signed integer (negative alignment scores are possible with global=0). aln-min-score also affects MAPQ estimates. The primary contributor to MAPQ calculation is the difference between the best and second-best alignment scores. A read's best alignment score is saved in the AS SAM tag, and the second-best score (if available) is saved in the XS tag. aln-min-score serves as the suboptimal alignment score if nothing higher was found except the best score. Therefore, increasing aln-min-score can decrease reported MAPQ for some low-scoring alignments. You can use the min-score-coeff option to adjust aln-min-score as a function of read length.

The pe-orientation option specifies the expected paired-end orientation. Only pairs with the orientation can be flagged as proper pairs. The following values are valid:

For paired-end reads, the best mapping positions are determined jointly for each pair. The positions are evaluated according to the largest pair score found, considering the various combinations of alignments for each mate. A pair score is the sum of the two alignment scores minus a pairing penalty, which estimates the unlikelihood of insert lengths further from the mean insert than this aligned pair.

The unpaired-pen option specifies how much alignment pair scores should be penalized when the two alignments are not in properly paired position or orientation. The option also serves as the maximum pairing penalty for properly paired alignments with extreme insert lengths.

The unpaired-pen option is specified in Phred scale, according to its potential impact on MAPQ. Internally, the penalty is scaled into the alignment score matrix based on Smith-Waterman scoring parameters.

The mapq-max option specifies a limit on the estimated MAPQ that can be reported for any alignment. Values from 0 to 255 are valid. If the calculated MAPQ is higher, the mapq-max value is reported instead. The default is 60.

The supp-aligns and sec-aligns options restrict the maximum number of supplementary (ie, chimeric and SAM FLAG 0x800) alignments and secondary (ie, suboptimal and SAM FLAG 0x100) alignments, respectively, that can be reported for each read. A maximum of 4095 supplementary alignments and 4095 secondary alignments can be reported for any read, in addition to a primary alignment. High settings for these two options impact speed so it is advisable to increase only as needed.

The sec-phred-delta option controls the secondary alignments that are emitted based on the alignment score relative to the primary reported alignment. Only secondary alignments within this Phred value of the primary are reported.

The sec-aligns-hard option suppresses the output of all secondary alignments if there are more secondary alignments than can be emitted. When not all secondary alignments can be output, set sec-aligns-hard to 1 to force the read to be unmapped.

When the supp-as-sec option is set to 1, then supplementary (chimeric) alignments are reported with SAM FLAG 0x100 instead of 0x800. The default value is 0. The supp-as-sec option provides compatibility with tools that do not support FLAG 0x800.