Novoalign Mismatches

[lwhitmore]

Hey I am trying to use novoalign however I need to set the mismatch rate to 3 per read . Is there any parameter that will do that with reads without quality scores. If not is there a way to do it with base quality scores.

Thanks
Leanne

[SNPsaurus]

I think if you give the format as fasta (-F FA) and set the align threshold to be 90 (-t 90) then it will only allow 3 mismatches. Or give mock quality scores and use the same threshold.

[lwhitmore]

Hey,

I have tried both and they have not worked its still outputting perfect alignments.

Any other ideas?

Thanks for your help,
Leanne

[SNPsaurus]

Could you give your command line and a snippet of the input file and what the output looks like? I was looking at some of my academic lab's scripts and this:
novoalign -d $repeat_index_file -r All -R 5 -F FA -f $fasta_file > $repeat_align_file
for example will give lots of mismatch alignments. Now, if I didn't use the -r All option it would return only the perfect alignments because I am aligning reads against an index of themselves, so the alignment finds itself and since that is the best it doesn't show any others. Don't know if the same situation might apply to you since this case is a little atypical (instead of aligning reads to a reference genome).

If you know reads with mismatches are in the input, what does it show for them in the output? Shouldn't it give either a U, NM or R and display some info for each read? Or is it filtering them away for some reason?

[lwhitmore]

Here is the command I am working with now
novoalign -r All -o SAM -d 1L1MdA_I_NovoInd_LW -f SRR519779test_NoQaul_LWv2.fa -F FA 1>pracv2.sam 2>pracv2.metrics


I am working with 2 reads
>SRR519779.1063095ILLUMINA-8787AF:52:FC:1:29:10379:5418length=36
TACCTTGACAGCAGAGTCTTGCCCAAC
>SRR519779.2989698ILLUMINA-8787AF:52:FC:1:81:1791:16837length=36
ACAGCAGAGTCTTGCCCAACACCCG

Now when I use the -t default setting and -t 90 I get the following sam output
SRR519779.1063095ILLUMINA-8787AF:52:FC:1:29:10379:5418length=36 0 L1MdA_I 441 24 27M * 0 0 TACCTTGACAGCAGAGTCTTGCCCAAC * PG:Z:novoalign AS:i:3 UQ:i:3 NM:i:1 MD:Z:7M19
SRR519779.2989698ILLUMINA-8787AF:52:FC:1:81:1791:16837length=36 0 L1MdA_I 448 24 25M * 0 0 ACAGCAGAGTCTTGCCCAACACCCG * PG:Z:novoalign AS:i:3 UQ:i:3 NM:i:1 MD:Z:0M24

Here is the sequence I am trying to align to

>Ref
CGACTCGAGACTCGAGCCCCGGGCTACCTTGCCAGCAGAGTCTTGCCCAACACCCGCAAGGGTCCACACGGGACTCCCCACGGGACCCTAAGACCTCTGGTGAGTGGATCACAGTGCCTGCCCCAATCCAATCGCGCGGAACTTGAGACTGCGGTACATAGGGAAGCAGGCTACCCGGGCCTGATCTGGGGCACAAGTCCCTTCCGCTCGACTCGWGACTCGAGCCCCGGGCTACCTTGCCAGCAGAGTCTTGCCCAACACCCGCAAGGGTCCACACGGGACTCCCCACGGGACCCTAAGACCTCTGGTGAGTGGATCACAGTGCCTGCCCCAATCCAATCGCGCGGAACTYGAGACTGCGGTACATAGGGAAGCAGGCTACCCGGGCCTGATCTGGGGCACAAGTCCCTTCCGCTCGACTCGAGACTCGAGCCCCGGGCTACCTTGMCAGCAGAGTCTTGCCCAACACCCGCAAGGGCCCACACGGGACTCCCCACGGGACCCTAAGACCTCTGGTGAGTGGAACACAGCGCCTACCCCAATCCAATCGCGTGGAACTTGAGACTGCGGTACATAGGGAAGCAGGCTACCCGGGCTTGATCTGGGGCACAAACCCCTTCCACTCCACTCGAGCCCCGGCTACCTTGCCAGCTGAGTCGCCTGACACCCGCAAGGGCCCACACAGGATTCCACACGTGATCCTAAGACCTCTAGTGAGTGGAACACAACTTCTGCCAGGAGTCTGGTTCGAACACCAGATATCTGGGTACCTGCCTTGCAAGAAGAGAGCTTGCCTGCAGAGAATACTCTGCCCACTGAAACTAAGGAGAGTGCTACCCTCCAGGTCTGCTCATAGAGGCTAACAGAGTCACCTGAAGAACAAGCTCTTAACAGTGACAACTAAAACAGCTAGCTTCAGAGATTACCAGATGGCGAAAGGCAAACGTAAGAATCCTACTAACAGAAATCAAGACCACTCACCATCATCAGAACGCAGCACTCCCACCCCACCTAGTCCTGGGCACCCCAACACAACCGAAAATCTAGACCCAGATTTAAAAACATTTCTCATGATGATGATAGAGGACATCAAGAAGGACTTTCATAAGTCACTTAAAGATTTACAGGAGAGCACTGCTAAAGAGTTACAGGCTCTTAAAGAAAAGCAGGAAAACACAGCCAAACAGGTGATGGAAATGAACAAAACCATACTAGAACTAAAAGGGGAAGTAGACACAATAAAGAAAACCCAAAGCGAGGCAACGCTGGAGATAGAAACCCTAGGAAAGAGATCTGGAACCATAGATGCGAGCATCAGCAACAGAATACAAGAAATGGAAGAGAGAATCTCAGGTGCAGAAGATTCCATAGAGAACATCGACACAACAGTCAAAGAAAATACAAAATGCAAAAGGATCCTAACTCAAAACATCCAGGTAATCCAGGACACAATGAGAAGACCAAACCTACGGATAATAGGAATTGATGAGAATGAAGATTTTCAACTTAAAGGGCCAGCTAATATCTTCAACAAAATAATAGAAGAAAACTTCCCAAACATAAAAAAAGAGATGCCCATGATCATACAAGAAGCATACAGAACTCCAAATAGACTGGACCAGAAAAGAAATTCCTCCCGACACATAATAATCAGAACAACAAATGCACTAAATAAAGATAGAATATTAAAAGCAGTAAGGGAGAAAGGTCAAGTAACATATAAAGGAAGGCCTATCAGAATTACACCAGACTTTTCACCAGAGACTATGAAAGCCAGAAGAGCCTGGACAGATGTTATACAGACACTAAGAGAACACAAATGCCAGCCCAGGCTACTATACCCGGCCAAACTCTCAATTACCATAGATGGAGAAACCAAAGTATTCCACGACAAAACCAAGTTCACACAATATCTTTCCACGAATCCAGCCCTTCAAAGGATAATAACAGAAAAGAAGCAATACAAGGACGGAAATCACGCCCTAGAACAACCAAGAAAGTAATCATTCAACAAACCAAAAAGAAGACAGCCACAAGAACAGAATGCCAACTCTAACAACAAAAATAAAAGGGAGCAACAATTACTTTTCCTTAATATCTCTTAATATCAATGGACTCAATTCCCCAATAAAAAGACATAGACTAACAGACTGGCTACACAAACAGGACCCAACATTCTGCTGCTTACAGGAAACCCATCTCAGGGAAAAAGACAGACACTACCTCAGAGTGAAAGGCTGGAAAACAATTTTCCAAGCAAATGGACTGAAGAAACAAGCTGGAGTAGCCATTTTAATATCGGATAAAATCGACTTCCAACCCAAAGTTATCAAAAAAGACAAGGAGGGACACTTCATACTCATCAAAGGTAAAATCCTCCAAGAGGAACTCTCAATTCTGAATATCTACGCACCAAATGCAAGGGCAGCCACATTCATTAGAGACACTTTAGTAAAGCTCAAAGCATACATTGCACCTCACACAATAATAGTGGGAGACTTCAACACACCACTTTCTTCAAAGGACAGATCGTGGAAACAGAAACTAAACAGGGACACAGTGAAACTAACAGAAGTTATGAAACAAATGGACCTGACAGATATCTACAGAACATTTTATCCTAAAACAAAAGGATATACCTTCTTCTCAGCACCTCACGGGACCTTCTCCAAAATTGACCATATAATTGGTCACAAAACAGGCCTCAATAGATACAAAAATATTGAAATTGTCCCATGTATCCTATCAGACCACCATGGCCTAAGACTGATCTTCAATAACAACATAAATAATGGAAAGCCAACATTCACGTGGAAACTGAATAACACTCTTCTCAATGATACCTTGGTCAAGGAAGGAATAAAGAAAGAAATTAAAGACTTTTTAGAGTTTAATGAAAATGAAGCCACAACGTACCCAAACCTATGGGACACAATGAAAGCATTTCTAAGAGGGAAACTCATAGCGCTGAGTGCCTCCAAGAAGAAACGGGAGACAGCACATACTAGCAGCTTGACAACACATCTAAAAGCCCTAGAAAAAAAGGAAGCAAATTCACCCAAGAGGAGTAGACGGCAGGAAATAATCAAACTCAGGGGTGAAATCAACCAAGTGGAAACAAGAAGAACTATTCAAAGAATTAACCAAACGAGGAGTTGGTTCTTTGAGAAAATCAACAAGATAGATAAACCCTTAGCTAGACTCACTAAAGGGCACAGGGACAAAATCCTAATTAACAAAATCAGAAATGAAAAGGGAGACATAACAACAGATCCTGAAGAAATCCAAAACACCATCAGATCCTTCTACAAAAGGCTATACTCAACAAAACTGGAAAACCTGGACGAAATGGACAAATTTCTGGACAGATACCAGGTACCAAAGTTGAATCAGGATCAAGTTGACCATCTAAACAGTCCCATATCACCTAAAGAAATAGAAGCAGTTATTAATAGTCTCCCAACCAAAAAAAGCCCAGGACCAGATGGGTTTAGTGCAGAGTTCTATCAGACCTTCAAAGAAGATCTAATTCCAATTCTGCACAAACTATTTCACAAAATAGAAGTAGAAGGTACTCTACCCAACTCATTTTATGAAGCCACTATTACTCTGATACCTAAACCACAGAAAGATCCAACAAAGATAGAGAACTTCAGACCAATTTCTCTTATGAATATCGATGCAAAAATCCTCAATAAAATTCTCGCTAACCGAATCCAAGAACACATTAAAGCAATCATCCATCCTGACCAAGTAGGTTTTATTCCAGGGATGCAGGGATGGTTTAATATACGAAAATCCATCAATGTAATCCATTATATAAACAAACTCAAAGACAAAAACCACATGATCATCTCGTTAGATGCAGAAAAAGCATTTGACAAGATCCAACACCCATTCATGATAAAAGTTTTGGAAAGATCAGGAATTCAAGGCCCATACCTAAACATGATAAAAGCAATCTACAGCAAACCAGTAGCCAACATCAAAGTAAATGGAGAGAAGCTGGAAGCAATCCCACTAAAATCAGGGACTAGACAAGGCTGCCCACTTTCTCCCTACCTTTTCAACATAGTACTTGAAGTATTAGCCAGAGCAATTCGACAACAAAAGGAGATCAAGGGGATACAAATTGGAAAAGAGGAAGTCAAAATATCACTTTTTGCAGATGATATGATAGTATATATAAGTGACCCTAAAAATTCTACCAGAGAACTCCTAAACCTGATAAACAGCTTCGGTGAAGTAGCTGGATATAAAATAAACTCAAACAAGTCAATGGCCTTTCTCTATACAAAGAATAAACAGGCTGAGAAAGAAATTAGGGAAACAACACCCTTCTCAATAGTCACAAATAATATAAAATATCTTGGCGTGACTCTAACTAAGGAGGTGAAAGATCTGTATGATAAAAACTTCAAATCTCTGAAGAAAGAAATTAAAGAAGATCTCAGAAGATGGAAAGATCTCCCATGCTCATGGATTGGCAGGATCAACATTGTAAAAATGGCTATCTTGCCAAAAGCAATCTACAGATTCAATGCAATCCCCATCAAAATTCCAACTCAATTCTTCAACGAATTGGAAGGAGCAATTTGCAAATTTGTCTGGAATAACAAAAAACCTAGGATAGCAAAAAGTCTTCTCAAGGATAAAAGAACTTCTGGCGGAATCACCATGCCAGACCTAAAGCTTTACTACAGAGCAATTGTGATAAAAACTGCATGGTACTGGTATAGAGACAGACAAGTAGACCAATGGAATAGAATTGAAGATCCAGAAATGAACCCACACACCTATGGTCACTTGATCTTCGACAAGGGAGCTAAAACCATCCAGTGGAAGAAAGACAGCATTTTCAACAATTGGTGCTGGCACAACTGGTTGTTATCGTGTAGAAGAATGCGAATCGATCCATACTTATCTCCTTGTACTAAGGTCAAATCTAAGTGGATCAAGGAACTTCACATAAAACCAGAGACACTGAAACTTATAGAGGAGAAAGTGGGGAAAAGCCTTGAAGATATGGGCACAGGGGAAAAATTCCTGAACAGAACAGCAATGGCTTGTGCTGTAAGATCGAGAATCGACAAATGGGACCTAATGAAACTCCAAAGTTTCTGCAAGGCAAAAGACACCGTCAATAAGACAAAAAGACCACCAACAGATTGGGAAAGGATCTTTACCTATCCTAAATCAGATAGGGGACTAATATCCAACATATATAAAGAACTCAAGAAGGTGGACTTCAGAAAATCAAATAACCCCATTAAAAAATGGGGCTCAGAACTGAACAAAGAATTCTCACCTGAGGAATACCGAATGGCAGAGAAGCACTTGAAAAAATGTTCAACATCCTTAATCATCAGGGAAATGCAAATCAAAACAACCCTGAGATTCCACCTCACACCAGTCAGAATGGCTAAGATCAAAAATTCAGGTGACAGCAGATGCTGGCGAGGATGTGGAGAAAGAGGAACACTCCTCCATTGTTGGTGGGAGTGCAGGCTTGTACAACCACTCTGGAAATCAGTCTGGCGGTTCCTCAGAAAACTGGACATAGTACTACCGGAGGATCCAGCAATACCTCTCCTGGGCATATATCCAGAAGATGCCCCAACAGGTAAGAAGGACACATGCTCCACTATGTTCATAGCAGCCTTATTTATAATAGCCAGAAGCTGGAAAGAACCTAGATGCCCCTCAACAGAGGAATGGATACAGAAAATGTGGTACATCTACACAATGGAGTACTACTCAGCTATTAAAAAGAATGAATTTATGAAATTCCTAGCCAAATGGATGGACCTGGAGGGCATCATCCTGAGTGAGGTAACACATTCACAAAGAAACTCACACAATATGTATTCACTGATAAGTGGATATTAGCCCCAAACCTAGGATACCCAAGATATAAGATATAATTTGCTAAACACATGAAACTCAAGGAGAATGAAGACTGAAGTGTGGACACTATGCCCCTCCTTAGATTTGGGAACAAAACACCCATGGAAGGAGTTACAGAGACGGAGTTTGGAGCTGAGATGAAAGGATGGACCATGTAGAGACTGCCATAGCCAGGGATCCACCCCATAATCAGCATCCAAACGCTGACACCATTGCATACACTAGCAAGATTTTATTGAAAGGACGCAGATGTAGCTGTCTCTTGTGAGACTATGCCGGGGCCCAGCAAACACAGAAGTGGATGCTCACAGTCAGCTAATGGATGGATCATAGGGCTCCCAATGGAGGAGCTAGAGAAAGTAGCCAAGGAGCTAAAGGGATCTGCAACCCTATAGGTGGAACAACATTATGAGCTAACCAGTACCCCGGAGCTCTTGACTCTAGCTGCATATATATCAAAAGATGGCCTAGTCGGCCATCACTGGAAAGAGAGGCCCATTGGACTTGCAAACTTTATATGCCCCAGTACAGGGGAATACCAGGGCCAAAAAGGGGGAGTGGGTGGGCAGGGGAGTGGGGGTGGGTGGATATGGGGGACTTTTGGTATAGCATTGGAAATGTAAATGAGTTAAATACCTAATAAAAAATGGAAAAAAA

[lwhitmore]

Just a note if there were 3 mismatches allowed I should be getting two more alignment results for the first read starting at bases 233 and 25

Thanks again

[SNPsaurus]

I'm a little hazy about this part, when does -r All stop reporting alignments? It must use some sort of cut-off. I would imagine for your short reads there would be a large jump in score between a perfect match and three mismatches so -t 90 might not be high enough. Try -E 10 and the -t option to the maximum score possible (depends on the novoalign version, I think). You could also try adding some extra reads that match the mismatch regions, match with 1 or 2 mismatches and see what they do. Sorry to be sort of guessing about this... it has been a while since I've played around in depth with novoalign.