Managing reference sequences

As part of a secondary analysis job, you can specify a reference sequence to align the SMRT Cells reads to so as to produce a consensus sequence. After you import a reference sequence, it becomes available for use with any protocol that requires a reference.

Note: You must be logged in as a scientist or administrator to import, edit, or delete reference sequences.

There are two different mechanisms for importing reference sequences: directly, and using the dropbox folder. Both methods require FASTA files.

To import a reference sequence directly

1. Click Design Job, then click Import and Manage.
2. Click Manage Reference Sequences, then click New.
3. Enter a name and organism for the sequence.
4. Choose a ploidy for the sequence.
5. Click Browse, then choose one or more FASTA file(s) to import. (Note: If a reference sequence is made up of multiple FASTA files, the files must be located in the same directory.)
6. Click Upload. The sequence is validated in the background. Depending on the complexity of the sequence, validation can take a few seconds to a few hours.

• If the sequence file passes validation, the imported sequence is added to the list. The state is set to Active, and the reference sequence is available for use with protocols. (You also receive an email message telling you that the sequence passed validation.)
• If the sequence file fails the validation process, you receive an email message with error information.  

To import a reference sequence using the dropbox folder

1. Copy one or more FASTA file(s) containing a reference sequence to $SEYMOUR_HOME/common/references_dropbox.

• Make sure that the smrtanalysis user has read permissions ls –l <reference.fasta>. The FASTA file(s) should have “r__r__r__” permissions.

2. Click Design Job, then click Import and Manage.
3. Click Manage Reference Sequences, then click Scan. A dialog displays reference sequence files located in the dropbox folder that are ready to be imported. (The location of the dropbox folder is set by the administrator.)
4. Select a sequence and click OK. The Reference Sequence Details dialog displays.
5. Enter a name and organism for the sequence.
6. Choose a ploidy for the sequence, then click OK. The sequence is validated in the background. Depending on the complexity of the sequence, validation can take a few seconds to a few hours.

• If the sequence file passes validation, the imported sequence is added to the list. The state is set to Active, and the reference sequence is available for use with protocols. (You also receive an email message telling you that the sequence passed validation.)
• If the sequence file fails the validation process, you receive an email message with error information.

To edit a reference sequence

1. Click Design Job, then click Import and Manage.
2. Click Manage Reference Sequences.
3. Select a reference sequence, then click Edit.
4. Edit the name, organism, ploidy, last modified date, and/or state. When the state is Active, the reference sequence is available for use with protocols.
5. Click OK.

To delete a reference sequence

1. Click Design Job, then Import and Manage.
2. Click Manage Reference Sequences, then select the reference sequence to delete.
3. Click Delete, then click OK in the confirmation dialog. The reference sequence is no longer available in SMRT Portal.