The cluster.fragments command groups sequences that are part of a larger sequence. To run this tutorial please download esophagus.fasta file
The cluster.fragments command expects a fasta-formatted file. A names file may also be provided and when a sequence is determined to be a fragment of a larger sequence, the list of names corresponding to the sequence names will be merged. Let’s take a look at the esophagus.fasta file. It contains 710 sequences.
mothur > unique.seqs(fasta=esophagus.fasta)
After running unique.seqs, we find that esophagus.unique.fasta contains 656 unique sequences. Now let’s see if any of those sequences are fragments of another sequence.
mothur > cluster.fragments(fasta=esophagus.unique.fasta, name=esophagus.names)
or with a count file:
mothur > cluster.fragments(fasta=esophagus.unique.fasta, count=esophagus.count_table)
After running cluster.fragments, we find that 22 sequences were fragments of larger sequences. Thus esophagus.unique.fragclust.fasta contains 634 unique sequences.
percent & diffs
The diffs parameter allows you to set the number of differences allowed, default=0. The percent parameter allows you to set percentage of differences allowed, default=0. percent=2 means if the number of difference is less than or equal to two percent of the length of the fragment, then cluster. You may use diffs and percent at the same time to say something like: If the number or differences is greater than 1 or more than 2% of the fragment length, don’t merge.
Explaining the output
Running the cluster.fragments command above will cause the following to be outputted to the screen:
0 656 0
100 643 13
200 639 17
300 638 18
400 635 21
500 634 22
600 634 22
656 634 22
This output indicates, by column, the number of sequences processed, the number of sequences that will be found in the final dataset, and the number of sequences that have been clustered away. This should accelerate as the function runs.
What’s the difference between cluster.fragments and pre.cluster?
- Cluster.fragments: combine sequences that are pieces of a larger sequence
- Pre.cluster: combine sequences that are within a certain number of diffs
Cluster.fragments was developed before pre.cluster. It has some of the same basic ideas but slight variations. Cluster.fragments processes unaligned sequences. It sorts them by sequence length and if there are ties in length then by abundance. It then goes through the sequences starting with the largest fragment as asks if each following sequence is a fragment of itself. You can set parameters to determine what this means, i.e. numdiffs, percentage differences. mothur does a pairwise alignment and counts the diffs merging fragments under the unique sequence. This process is all very much like pre.cluster.
Pre.cluster sorts by abundance only to determine the order. Pre.cluster allows for clustering within samples, cluster.fragments does not. Pre.cluster is parallelized. Pre.cluster works with both aligned and unaligned sequences. Pre.cluster only uses diffs not percentages. Pre.cluster allows you to set alignment methods and parameters for unaligned sequences, cluster.fragments does not.
We use pre.cluster in our lab. The distinguishing feature to cluster.fragments is the ability to sort unaligned sequences by length.
- 1.28.0 Added count parameter