Axe’s matching algorithm

Axe uses an algorithm based on longest-prefix-in-trie matching to match a variable length from the start of each read against a set of ‘mutated’ barcodes.

Hamming distance matching

While for most applications in high-throughput sequencing hamming distances are a frowned-upon metric, it is typical for HTS read barcodes to be designed to tolerate a certain level of hamming mismatches. Given these sequences are short and typically occur at the 5’ end of reads, insertions and deletions rarely need be considered, and the increased rate of assignment of reads with many errors is offset by the risk of falsely assigning barcodes to an incorrect sample. In any case, reads with more than 1-2 sequencing errors in their first several bases are likely to be poor quality, and will simply be filtered out during downstream quality control.

Hamming mismatch tries

Typically, reads are matched to a set of barcodes by calculating the hamming distance between the barcode, and the first l bases of a read for a barcode of length l. The “correct” barcode is then selected by recording either the barcode with the lowest hamming distance to the read (competitive matching) or by simply accepting the first barcode with a hamming distance below a certain threshold. These approaches are both very computationally expensive, and can have lower accuracy than the algorithm I propose. Additionally, implementations of these methods rarely handle barcodes of differing length and combinatorial barcoding well, if at all.

Central to Axe’s algorithm is the concept of hamming-mismatch tries. A trie is a N-ary tree for an N letter alphabet. In the case of high-throughput sequencing reads, we have the alphabet AGCT, corresponding to the four nucleotides of DNA, plus N, used to represent ambiguous base calls. Instead of matching each barcode to each read, we pre-calculate all allowable sequences at each mismatch level, and store these in level-wise tries. For example, to match to a hamming distance of 2, we create three tries: One containing all barcodes, verbatim, and two tries where every sequence within a hamming distance of 1 and 2 of each barcode respectively. Hereafter, these tries are referred to as the 0, 1 and 2-mm tries, for a hamming distance (mismatch) of 0, 1 and 2. Then, we find the longest prefix in each sequence read in the 0mm trie. If this prefix is not a valid leaf in the 0mm trie, we find the longest prefix in the 1mm trie, and so on for all tries in ascending order. If no prefix of the read is a complete sequence in any trie, the read is assigned to an “non-barcoded” output file.

This algorithm ensures optimal barcode matching in many ways, but is also extremely fast. In situations with barcodes of differing length, we ensure that the longest acceptable barcode at a given hamming distance is chosen; assuming that sequence is random after the barcode, the probability of false assignments using this method is low. We also ensure that short perfect matches are preferred to longer inexact matches, as we firstly only consider barcodes with no error, then 1 error, and so on. This ensures that reads with barcodes that are followed by random sequence that happens to inexactly match a longer barcode in the set are not falsely assigned to this longer barcode.

The speed of this algorithm is largely due to the constant time matching algorithm with respect to the number of barcodes to match. The time taken to match each read is proportional instead to the length of the barcodes, as for a barcode of length l, at most l + 1 trie level descents are required to find an entry in the trie. As this length is more-or-less constant and small, the overall complexity of axe’s algorithm is O(n) for n reads, as opposed to O(nm) for n reads and m barcodes as is typical for traditional matching algorithms