Sequence Alignment Protocols

Aligning sequences using MAFFT (Multiple Alignment using Fast Fourier Transform)

MAFFT is a multiple sequence alignment tool available for online job submissions or for download into a PC or workstation. In the online version, a result page with a URL similar to https://mafft.cbrc.jp/alignment/server/spool/_ho.210128090610553ohuTlbaK9CXQxxxxx.html can be accessed by anyone who knows this URL. The input files and results are temporarily stored in the MAFFT server and deleted after 96 hours of receiving the email with your alignment results. Alternatively, for strictly confidential data, it is recommended to download MAFFT onto your computer.

Quick guide on how to use the online version of MAFTT

1. Access the online MAFFT tool and follow instructions in the website https://mafft.cbrc.jp/alignment/server/add_fragments.html
2. Upload the reference dataset in .txt format into the "Existing alignment" box
3. Upload the test dataset in .txt format into the "Fragmentary sequence(s)" box
4. Check box Allow unusual symbols (Selenocysteine "U", Inosine "i", non-alphabetical characters, etc.)
5. Provide a job name and email address to get results by email
6. Change the following parameters in "Advanced Settings" for the MAFFT alignment:

• "Remove uninformative sequences": select 100% (Nothing removed) or adjust to remove sequences that have ambiguous letters
• "Keep alignment length": select Yes to delete insertions in the test dataset to keep the alignment length the same as the reference alignment
• "Strategy": select Auto
• "Parameters": select Scoring matrix for nucleotide sequences: 200PAM; select (nwildcard) N is treated like a wildcard (this option keeps "N" runs aligned)

7. MAFFT will send email with results within one hour of job submission (depending on server status)
8. Save alignment as a FASTA file

Quick guide on how to download and run MAFTT in your computer

Download MAFFT 7 (version 7.475, 2020/Nov) from the MAFFT-CBRC website and follow the installation instructions (https://mafft.cbrc.jp/alignment/software/).

These steps were done using a PC with the following description:

Manufacturer: Dell Inc.

PC Model: Latitude 7400 2-in-1 Serial N Total RAM: 16127 MB

Drive C Capacity: 487246 MB (77% free)

1. Download the MAFFT zip folder and follow the installation instructions (use the All-in-one package for Windows)
2. Save the sequences in FASTA format
3. Copy the FASTA file into the same folder where the mafft.bat file is located
4. Open the MAFFT console
5. Write the FASTA filename for the input file option (Copy/Paste function works and add file extension .fas, .fasta or .txt) and press ENTER
6. Write new FASTA filename and add .fasta extension for the output file option, press ENTER
7. Write “3” to select the output format Fasta format / Sorted (saves aligned sequences in sorted order and FASTA format) and press ENTER
8. Write “1” to select --auto strategy (MAFFT will choose the best alignment option) and press ENTER
9. Write "--thread -1" in the argument option (if needed, a list of additional arguments can be found here: https://mafft.cbrc.jp/alignment/software/manual/manual.html#lbAI)

Note: Before loading FASTA alignments into HantaNet, inspect the MAFFT alignment using an alignment editor to confirm that new sequences were aligned correctly against the reference alignment available in HantaNet.