Align.seqs (Warning)

I did alignment of my sequence with the reference database using the following command
align.seqs(fasta=stability.trim.contigs.unique.fasta, reference=silva.v4.fasta)
As a result I got this Warning and a Note

Can someone please explain what these mean? And Do i need to do any correction after this step?

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Hello! It means a lot of sequence have been discarded because they could not align correctly.

It might be because the region you are trying to align t is not correct or that for a lot of reads you need to use the reverse-complemented for the alignment. If you are using the latest version, 1.47, this option is by default set to true, but in your case, from the message, it seems you are not using the latest version, so you need to upgrade to the latest version (preferable) or add in the command “flip=t”.

Best of success

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Thank you for replying.
I am using the latest version 1.47. on macbook.

I did add “flip=t” in the command and still got that warning.
Details Given below:
mothur > align.seqs(fasta=stability.trim.contigs.unique.fasta, reference=silva.v4.fasta, flip=t)

Using 8 processors.

Reading in the /users/hiraabid/desktop/mothur/M06339_Run_192-Semrau_Soil_Microcosm/silva.v4.fasta template sequences… DONE.
It took 3 to read 14956 sequences.

Aligning sequences from /users/hiraabid/desktop/mothur/M06339_Run_192-Semrau_Soil_Microcosm/stability.trim.contigs.unique.fasta …

It took 426 secs to align 1283076 sequences.

[WARNING]: 1352 of your sequences generated alignments that eliminated too many bases, a list is provided in /users/hiraabid/desktop/mothur/M06339_Run_192-Semrau_Soil_Microcosm/stability.trim.contigs.unique.flip.accnos.
[NOTE]: 261 of your sequences were reversed to produce a better alignment.

It took 426 seconds to align 1283076 sequences.

Output File Names:


This is not a lot of sequences. They are most probably errors that you do not want to have in your file. You can proceed with the analysis and ignore this warning.

Best of success

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Thank you for replying :slight_smile:

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