PacBio fastq and VICUNA

[Mesmer]

Hi, we have PacBio data for a De Novo assembly project of viral sequences. The run was made in long reads.
I analyzed the data with smrtpipe LongAmpliconAnalysis. The config file follows :

<?xml version="1.0"?>
<smrtpipeSettings>
   <module name="P_Fetch"/>
   <module name="P_Filter"/>
   <module name="P_Barcode">
     <param name="barcode.fasta">
       <value>barcode_run1.fasta</value>
     </param>
     <param name="mode">
       <value>symmetric</value>
     </param>
     <param name="adapterSidePad">
       <value>2</value>
     </param>
     <param name="insertSidePad">
       <value>2</value>
     </param>
   </module>
   <module name="P_AmpliconAnalysis">
	<param name="minLength">
       <value>2400</value>
     </param>
	 <param name="minReadScore">
       <value>0.78</value>
     </param>
     <param name="maxReads">
       <value>700</value>
     </param>
   </module>
</smrtpipeSettings>

The consensus generated by this pipeline are allright for almost all samples, we manage to create contigs of the length of the expected genome using MIRA with the resulting fastqs or manually with the fastas.

But we wanted to give a try with VICUNA to extract quasi-species information. When I tried to launch VICUNA on the fastq resulting from the demultiplexing step (barcoded-fasqs folder):

vicuna vicuna_config_11F1.txt 

--------------------------------------------------------
Program runs with the following Parameter setting:

	===== Trimmer =====

	vectorFileName	
	trimLogFileName	
	minMSize	9
	minInternalMSize	15
	maxOverhangSize	4
	minReadSize	25

	===== Profiler =====

	MSAFileName	
	binNumber	20
	kmerLength	15 (encode using 4 bytes)
	maxHD	1
	minSpan	75
	blockNumber	5
	rMapFileName	

	===== Contiger =====

	w1	12
	w2	5
	Divergence	8
	max_read_overhang	2
	min_profile_col_weight	5
	min_consensus_base_ratio	85
	max_contig_overhang	10
	seed_kmer_len	12
	min_contig_overlap	25
	min_contig_links	3
	min_identity	90
	min_perc_polymorphism	5
	max_variant_len	20

	===== Assembly =====

	npFqDir	/home/nico/labo/etudes/HEPAC/run_1/deNovo_VICUNA/data
	batchSize	2000000
	LibSizeLowerBound	100
	LibSizeUpperBound	800
	min_output_contig_len	300
	outputDIR	/home/nico/labo/etudes/HEPAC/run_1/deNovo_VICUNA/11F1/

--------------------------------------------------------

Indexing ...

	/home/nico/labo/etudes/HEPAC/run_1/deNovo_VICUNA/data/11F1_lbc11--lbc11--11F1_lbc11--lbc11.fastq
create: err reading fastq file:
/home/nico/labo/etudes/HEPAC/run_1/deNovo_VICUNA/data/11F1_lbc11--lbc11--11F1_lbc11--lbc11.fastq ... exit

VICUNA can't read the fastq and the error is a little fuzzy. I tried with fastq originating from MiSeq sequencing and it works fine.

fastq from PacBio:

@m160321_175808_42263_c100986322550000001823225107191690_s1_X0/32876/1557_2547 0.87 28
AACTTAAGATAGTTGGTGACCATCCGCTGGTGATAGAGCGTGTGCGGGCCTATTGCTGCCACTTTTGTGCTGCTGCTCCACTGCGGCCCCTGAGCCGTCACCAATGCTTATGTCCCATACCCCCGTCGACAAGGTGTATGTTCGTTCACATATTTGGCCCTGGCGGGTCCGCCGTCCCTGTTCCATCAGCCTGCTCTACAAAAATCCACATTTCATGCGTCCCAGTTTCAATATTTGGGACCGGGCTCATGCTCATTTGGTGCCCCCCTGGACGATCAGTGCGTTTTGCTGCTCACGGCTTTTTATGACTTACCTTCGTGGATTGTTACAAGGTTACTGTAGGTGCCCTTGTTGTTCCCTAATGAAGGGTGGAATGCTTCGGAGGAAACGCTGCTCACCCTGTTACACCGCAGCGTACTTGACCATTTTCATCAGCGTTAGCCTCCGTACTCAAAGCTATATCCAAGGCATGCGCCGGCTGGAGGTTGAGCAATGCTCAAAAAATTTATCACAAAAGACCTATAGTTGGCTGTTTTGAGAAGTCTGGCCGTGACTACATCCCCGGCCGTCAGCTTTCAGTTCTATGCACAGTGCCGCCGCTGGCTATCGGCGGGTTTCCACCTCGATCAAGGGTGCCTGTTTTTTTGATGAGTCTGCTCCCTGCCGTTTGTAGGCGTTTCTTAGAAAGGTTTGCAGGTAAGTTCTGCTGTTTTTATGAAGTGGCTGGGGCCAGGAGTGCACCTGCTTTCCTTGGAAACGCAGCTGAGGGCCTGGTTGGTGACCATGCCACGATAATGAAGCTATGGAGGGTTCTGAGGTCGACCAGGCTGAGCCCGCTCATCTTGATGTTTTCTGGGACTTATGGCCGTCCAATGGAAGCCAACCTCGAGGCCCTGTACAGGGCGCTTAACATCCCGCACGATATCGCCGCTTTGGGGCCTCCATGGCGTCGACATCCGC
+
(.(,'(..+-$-,,,-..-++,+#+,-.-.///./.(.-+/./%/)('%/./+.-/)-,,//.++-//)/+.///.+*./-'-.+*$%(..)//,//,//,/)//./%///--&&)///*)$)/-///-.(&./////'/*.//-/.#///*.+-.,$*/+.+/#%+-%-%+/))'++//-/+////&.+/,/++....$%*(-+*/////*///*.$(/',///'*./%/-/&,-,--.).*+,.)//*/.//#.+*-./.#(*$$(.--/+//////%./*-&%-*/..+/..,',/,')*///',/.//,+*+()#*+,+%!*')/%/..*.//+/.,&-.-%-''-.-/(#(').()/(.*/--./.)*//./&./**--)'#.///.$////*+///-//&.+/.+///*//..,./-,//&+),&./)*//-(.$+).%.).)-//%$/*.,////,/*/.)//(../,//&/',+/.+-+//./$///./+,,'.&//'//////-$%.+/,//.-//.-.-/.+.,'.--+).+/,..,&/-///////(/))+..,+//,,)//&)./..*&&''/-/*//,/.+/-,/-/*-(/.-.(.,/,,--)/+//'.//../)*/)',+.).,..#*#+*../.+/%/////-)-&/-*/+,#,--'+*&,+&/.-//%(*/..,+..//.-,*&*'////&.//*+*,*..),(.&+//,*-*+.//-///./),///..*/*/(-**%'/+$//,//./,*)$/.-/*,-///*,/./.,,/+//+*///)..*+#+*&-',.,/,///*.//./)/+/-//.//),//././.+.-.+//.+$///,+-(.....-#(/-.-+(-.(*$..+.).''.....)(&/*././/--+*//-..#...).(+-./*//.//*.'*+,$,*),$%.++/+..*/*-/.(.+(),./

fastq from MiSeq:

@MISEQ3:10:000000000-AGH1Y:1:1101:19170:2650 1:N:0:CGAGGCTGACTGCATA
GACCATCAGCGGCCCGAAAGGACCGGTCAACCAGATGTACACCAATGTCGACCAAGACTTGGTGGGTTGGCCCGCACCTCCAGGAGTGAAGTCCTTGGCCCCATGCACCTGTGGCTCGTCGGACCTGTTCCTGGTTACCAGGCACGCCGACGTGGTGCCCGTGCGCAGAAGAGGCGACACTCGTGGCGCCCTCCTAAGCCCCAGGCCAATTTCAACTCTTAAGGGGTCGTCCGGTGGGCCACTGCTGTG
+
CDDDDFFFFFDDGGGGGGGGGGHHGGGGGHHHGHHHHHHHHHHHHHHHHHHGGGHHHHHHHHHGGGGGGGHHHGGGGGHHHHHHHGGHHHHHHHHHHGHHHGGHHHHHHHHHHHHHHGGG@DDGGFB=FDGHFHHHHHHH0CGEGDGGGGGGBFGCEFGGGAD?B@?@;FFFFFFFCCF;0BAFEFFFFFFFFFFFFBFFFFFBFFFAFBFFF0FBBFBFFFFFFFFFFFFFFF-@FDF?FFFFEFFFF

Any clue?

[GenoMax]

FYI: I moved your thread to PacBio forum since folks from PacBio check that regularly.

You appear to have -- (hyphens) in your file names. Wonder if Vicuna is trying to interprete those. Have you tried changing those to _ ? Just a guess.

[rhall]

I don't know anything about Vicuna, but some applications can have problems with "/" or " " in fastq headers, I would test to see what happens if you simplify the fastq headers:

cat input.fastq | awk '/^@/{print "@" ++i; next}{print}'>output.fastq

[Mesmer]

Thanks guys for the help, I tried your solutions but no better results. But it makes me go deeper into the fastq files and I found that some entries were empty:

@m160321_175808_42263_c100986322550000001823225107191690_s1_X0/33015/0_3142 0.89 27
GCTGGAGCGCGGACCACAACATCAACGTCCCCTGCTGTATAGACCGCGACTTCCCTGAACCTGGAGACCGGATCCTGCCGGCGGTAAATTGGTAGTGCACAACCCCAGGGCTAACTGTAACACCGGCACAAGCCCGGCCAACCTCCGTGCAGCGTCGATCTCTAAGTGCTGGTTGTTCTGTACGGGCTGTATCTTCAGTTATTGTAGGGCCGGTTGTGCTGGCTTGTTGGCCTCGAAACCAGGTAGCGGCGAGGTCGGTCAAAGTAAAAGCTCATCCCCGGGGCGATGTTAACGCTCCCAAGCGTCAAAGCTGAGGCTAACGGGAACTCGGTAATATACGTGACCGAAGAAGTGGGTAAGCGGCGGTGCCACGGCGGGAGGCAGGTCTCTCGATATGCCTGCCATCAAGCCTCTTTCGGGTTCTGCTAACCTAATACTGCGGAGCCAACCGCATGGATAATAGGCCGAGGAGGACAGAAGTATATGCGGAAGACCATCACGCATAAGAACTCATTGGGTGGAACGAACTCGGGGTAGCGTTTGGTAAAAAGCATGGCAAAGGCCGCTCCGGGACGATGGCGGGATTCGACGCATGTGACCAGCCGAATCACAGTCTGACTCAAACAGTGACCCCGCATACACCTTCGCCCCATCCGGATAAGTGGTAGAGGAGGCGGCGGGTACGCGGAGGTGGTGGTGGTCGGTGGCTTTAACACGGGGCCTTCGGCTAGCAGGCCGGGCGGTTGAACGGCTCAGGGACTGAGGAGCAGGCGCAGCATTCAACCTGGACCTCAGAGGGTGGCGGGCAACACGCAAATGTCACTAACAAGGGGTTGTAGGGTGGAGCAACCCTGTGGCAGCGCCGGGCACAGCGGCAGCCGCTCAGGGGGGGAAAAATCACTAGAAAAACCAGATGTTGGACGGCAAGTCCCGGGTGTACAAAGTTCAATCCCCACAAAAAGGATTAGCGGACTCCCAGAGAGCCCGGGGGAAAAGGGGGTGGGAAGATGCCCAATAGCCCTCAGGATGCAGCCCAGACCGCCTGTAAGCGAATGTCGCTGGTGTAACCTGTTTGTACTATAGAGGGCACTGCAAAAAGCTGCTACCTCCCCGGAGCCTGCACTAGGGGCTCCCGCCCGGGGGAATGGTTGCAGTGCAATCCAGGCCATTAATGAAATCTTAACCTGCAAACGCAGCAGGAGTGAGTTTCATAGGTGAGGTTTATGCGGCCCGGCCCCCAATAGAGCTGGCAGGAAGCGTCGAACGAAAGGACATACTGCTCGGGGCCATTCGCCTCAAGATGGCGCATCAACCACCGTTGTCCGGAAGGTCTATTCCCGAGCACAGTGCGACACCTCAAACGGTCGGACCTGCCCAGTGAGTTCAAGCAGTGGCAGTTTAAACGGGAGGCTCTGAGCGGCGATATGCGTGGCGGGATGTTAGCGCCCTGTACAGGGCCTCGAGTTGGCTCCCATTGGACGGCATAAGTCCGCAGAGACATCAAGATGAGCGGGCTCGGCCCTGGTCGACGCTCAGAAACCCTCATAGTGCTTCAATTATCGTGGCCATGGTCACCAAACCAGGCCCTCAGTCTGGTTCCAGGAAGCAGGTGCACTCAGCCCAGCCACTTCTAAACAGCAGAACTTACCTGCAACCTTCTTAAGAACGTCCTACAACGGCAGGGAGCAGACTTCATCAAACAAGCACCCTTGGATCGAGGTGGAAACGCCGCGATAGCCAGCGGCGGCACTGTGCATAGAACTGAAGCTGACGGCCGGGGATGTAGTCACGGCCAGACTTCTCAAACAGCATCAACTATAGAGTCTTGTGATAAATTTTGGAGCATGCTCACTCCAGCCGGCGATAGACCCTTGGAATATAGCTTGAGTTACGGAAGGTAACGCTGATGACAAATGGTCAGTACGCTGCGGTGATAACAGCGGTGAGAGCGTCCTCCGAAGCATTCCACCATTCATTAGCAACAAGGGCACCTACAGTAACCTTGTAACTAATCCGCACGAAAGGTAAGTCATAAGCCGTGAGCCGCAAACGCCTGATCGTCCAGGGTGGCACCAAAGAGCATGAGCCGGTCCAAATATGAATGGGGACGGCATGAATTGGTGGTTTTGTAGAGCAAGCTGATGGAAACAGGGACGGGGACCCGCCCAGGGCCAAATATGGAACGAACATACAACCTCTGTCGACCGGGGGTATGGGACGTGATCAGGCATTGTGACGGCTCAGGGGCCGCAGTGAGGCAGACAGCACAAAGTGGCAGCCAATAGCCGCACACGCCTCTATCCACGCAGCCGGATGGTCACAGCTAACTATCTTAGTTGTGCGGATCCATGCGCGGAGTATAGAAAATCATGGTTGTAACCTGCTACTAGTGTCACCCCTCATAAGTCACAACCGGCACGTATCGCCGTCGTGAATAAGAAGGTATAGGTTGTGTGTAAGTGCCAGGTTGGTAGTAATACCTTCAGGGGGGAGATGTAGTGAGCAATATAAGCGTTGTCATGCCATGTCGGGCCATGGCTCTGCAAACATCGGCCGCCAGAGGTCATGGGCAATGAATAGAGGGCAACACCAGTCTCCCGCAGCGGAACGAGCAAACGAGAGAATCATCAAAACAGTAGGTGCGGTCAAAACGGGTGGGAGCCGCGCAAAGCGGAGCGGCGGCAGTTGGCCGCAGGGCCACGGGTAGGGGCAGAGTACCAGCGCTGGAACATCTCTGCCAACTGGTCGTAGGAAAGCAGCGATGTAAAACATTCGGTTGTCATTGATGGATCTTTGGGTGGGCCCCAACCTCTAAAAGCGACCAGCCCGGGCGCCGGCAAGTACTGTTCTTTTTAACTCATTATGAATAACCCGTGAATAGGATGATTCCAGAGCAGCTTCAGGTCGGAAACCAGCTGCGCGGGGTTTGCAATCAAATTAATAAGAATCTCAGTTTGGCACACGAGATAAAAACGGCCGAACCACCCACAGCATTTCGCACAGGGCAGAATTGGCCGCAGCCAGAGCAGCTTGCTCAATGGCAGTAGTGCTTGGGGATGCCAGGAGCATTAAAATCGAACTGATGGGCCCCTCCATGGCATCGACCACATGC
+
%,,$,--..*.+.,/-.*//-/.()...%&)'*..+//+/-.,)&%-,-),.(,'..*(*.-++#+((+#"#"((#"$-,(/./+,*/+.-..*//./*.)/*)*..,,(,.)//%..$$(&-)+,//.)/.-)/-(++)/)$+&&$,#(../-+.,/-//.-%*).)+)(,(*$%)*&--+(+-).+.../.-.%.().+-*..-+,*&...#,..)..-'),'.--.,(.///'&))/...--.,*&/.,..//(../,*///(,)////.)/))*.)(-+//&//-,*///.*)'/*/,///+,*////*//-/,*+/,-+&/.//)//%+/...*&--,%,.$-.*/*.%+./&(+,.,/../-#...-(++)+./&/....+../*./(//+%/..).#..)..&-+.#&-/,,.+,&.-..$/+./$*.,#"#'*'-+%-**$-(/&-,-..,##.&**/--+,.-*#'-/%/,.,&.-,'*((-.-(/....',()-*./)/*///'**,.,-.++..-*,./,+,.//+/-.&+-...#,))..//.,),++.)+/,./.+,--/////*,/,,-/)/////.///$/.-%'#,).(,#////+//.+*.-//,*//////)*,(%..+.///#--/,/**+.//,/..//%///$,././.-./,//+-$-+/)/./../..,)'--..(.//).,-++()-+&,.-,*-+*/)///+/..////,,/'-#/+,,.-*/*.*.('*-,-///.-.'/'//--$.-*///*..*/+./.&.)(/./..,,./',/#,$(&-&.(&++)///,(./'*/.*.)*(--)/./,-..--($&)/*,///.&,,.//.,/-'(////.//%*..,//-&..-)$'')++,*(*/////./.*,(/-/,//-/-,++./%$/)/..'+/,-.,.*-*,*..,/.##-()*...,,+)//./-//...-...(,%**../&(+-('#(,%+(-+$$)+%*,.*////)),'(/'..)++//-/.+-./*/%+...&)-/*///.*/.//%)'//.,--,-$..+)&.-..&-...('--...+-+.(//-,,,,)/////-,-%+%%%%---./*/,///./.,*-+.,&&-.$'-,*),.*//*//+././.,.)*-%-./+.)'./+(//)&///./%/,&(.**)/.%+)./..)///./-.)////.)././.+.'//,.+*+(+..,()'%-#-/../#,..#//-**/...//.)//.)*..-.)&-../(.(/+*(*+//-//.&),.)-+,,('##"',-&/+//,//,-.'&*#('+,+)*,-#-'+/*-(..//./-(....&+,+*($#")()--)+/+**-"/+-,/&,/)*/&.//--,//-.%.'$/.,-,,*-.+/#,.**-(/.//)/%///.%.,,.%..).-$/.),////./*(-**///-//.-.-'/(%//*.-.//.,/&/+///*/$).,*+/////$+,.//,'/,.,././,(+//,*.//*($.///.()/),(/'/+///..-//$..//////-)+/*-./)//,/('/,/$/,)+--.//'.)./,/+/..-)///'//.-.-./.+,$$,#(*+.,()&%%#../////*//.//+/,./)/+/+,/./-',*(-//#/...)//.)/.--,)././/-$///*/+)-/"-../%+-&--.///.+.-/.-,)/*$,///./+/*,////.)-/,/.*///-+///'*////*///-/.//,,/-*,.////,//.///*+/////--../$+&-.-"$'/$-/-.,/./....-..)//&%.+#)--,...//,-/.&./+../*/.,+.).#'#'*..---$-//*./.-*..-'//...%*.//)/////,/*/./,$.(../*,/.//..--..*.*//*/'.$/-..,--/)..')/++/.*//.%-/*,-)/%-,//..-..$/.*/,-*/.+/../+/,%.,&-'//*///)//,/%////$*...,*/&.,)/*/+.)./.---##('&./.)//////.&*/.-*-..&/.'/,*/..*.//.,+'/.-.(,,)+///,'&$)((+.//,-///*,).#..+*&$)*,.../#**//.//+/.-*-'./-&,-.,+$)$)&--%+/)--++/,*////*.%///*//////%.+.///////.+%')(&.//-,,,..(-$(-$&#+.$..-/(.//-.//.-*-,#/-////./,$/./(///)/.'+)//++//.,/*/,..'+.)..%**)-'.../$+//"///,/..//./,...%#.#)//++/..///..//./..//*/*/)/./.+.///+/,,,&/////.((-$)(.+/)..%..+/.../).,&*,/-/,*////.+(.).-)..+/(.+/.,/..////&),*//$....).,%)&*')(,'.)....)....,,*....-,$..%.,..-,'&'+*...,...*.-().(....(.-,-)&-.*./%*.../).,-*+*//.+,//...,$.+*..-,+,.+//*).'/../-$$/*///-'/)'.+,,')///,///.'%(,$-.$..).*//.,(&%)//././/*),/./(+,)*/&./*--*%#&$-.+#)//,)-/+,,(.++,/./,*.,/-.///.,./,/./.+-*+/.//--+-#//(+%..,*,)..&*./',/.,////,(&-)///////,-)//,./..//*..+&,////-.).$'(&'.//*//-/-.////..//,...//.'#*,./+-+(),)).,.+-.),),(./.+/.&-(.,++/$.*/'-%///.&//,/.&##$.*/////.//-/*/.*/*(/+./*///..////-/'-+//.//&/*///(/...--*,&+-$/./.$/,&+..%../%/,/,*/-.+/,'//*///%./+.'+.%//..//////&,,)//.,.'/),.).(-),-,(./-$/'./)/.&*(/+-*/*-.++/.+//+./,/.*///....//)/.+)/#-+(..-($'$"$.)//,//*-/./$./)*#-/%/)/-'/.*'+)'$),.(.#%*(./)/.)/&+/..%.
[B]@m160321_175808_42263_c100986322550000001823225107191690_s1_X0/33015/3183_3199 0.89 27

+

[/B]@m160321_175808_42263_c100986322550000001823225107191690_s1_X0/33015/3240_6404 0.89 27
CTCTTCTTTTCCTCCTCCTCCGTTGTTGTTGTTGAGAGAGATCGATCAGCTGAGGCGCGATGTGGTCGATGCCATGGAGGCCGCATCAGTTCATTAAGGCTCTGGCATCACTACTGCCAATTGAGCAAGCTGCTCTGGCTGCGGCCATTCTGCCCTGGCGAATGCCTGGTGGTGGTTCGGCCGTTTTTATCTGCGTGGGTGGCAACTGAGATTCTTATATTTATGCACCCCCGGCAGCTGGTTTTCCGACCTGAAGTGCTCTGGAATCATCCTATCCAACGGGTTATTCATAATGAGTTTAGAACAGTACTTGCCGGGCCCGGGCTGTCGCTGTTTTAGAGGGTTGGGGGCCCACCCAGATCGCATCAATGACAACCCGAATGTTTTACATCGCTGCTTCCTACGCAGTTGGCAGAGATTTGTCAGCGCTGGTACTCTGCCCTACCCGTGGCCCTCGGCCAACTGCCGCCGCTCGCTTTGCGCGGCTCCCCCCGTTTGACCGCACCTATGTTTTGATGGATTCTTCGTTGCTCGTTCGCTGCGGAAGACTGGTGTTGCCCTCTATTTCATTGCCATGAGCCTCCTGCCCGGCCGATGTGCCAGAGGCCCATGGCCCGAACATGGGATGACACGCTTATATGCTGCACTACATCTGCCCCTGAAGTAATTACTACCACCTTGGCACTTACAACAACTTCAATACCTTCTTATTCACGACGGCGATCGTGCGTTGTGAATTATGAGGGCGACACTAGTGCAGGTACAACATGATGTTTCTATACCTCCGCGCATGGATCCGCACACTAAGATAGTTTGGTGACCATTCCGCTGGTGATAGAGCGTGTGCAGGGCTATGGCTGCCACTTTGTGCTGCTGCTCACTGCGGCCCCCCATGAGCGTCACCAAATGGCCCTTATGTCCCATACCCCGGTCGACAGAGGTGTATGTTCGTTCCATATTTGGCCCTGGCGGTCCCCGTCCTGTTTCCCATCAGCCTGCTCTACAAAATCACATTTCTATTTGGCCGCCGGTTCATATTTGGGACCGTGCTCATGCTCATGTTGGGTGGGCACCTGGACGATCAGGCGTTTTTGGCTGCTCACGGCTTATGAAATACCTTGTGGGATTAAGTTTTTACCAAGGTTTACTGTTAGGTGGCCCTTGTTGGCTAATGAAGGGTGGAATGCTTCGGAGGACGCTCTTCACCGCTGTTATCACCGCAGCGTACTTGACCATTGTCATCAAGCGTTACCTCCGTACTCAAGCTATATCCCAGGCATGCGCCGGCTGGAGGTTGAGCATGCTCAAAATTTTCAACAAGACTCTATAGTTGGCTGTTTGAGAAGTCTGGCCGTGACTACATCCCGGCCGTCGTTCAGTTCTATGCACAGCGCGCCGCTGGCTATCGCGGGTTTCCACCTCGATCCAAGGGTGCTTGTTTTTGAATGAGTCTGCTCACTGCCGTTGTAGGACTTTCTTAAGAAGGTTGCTAGGTAAGTTCTGCTTGTTTTATGAAGTGGCTGGGGCATGGAGTGAGCCTGCTTCCTGGAACCAGCTGAGGGCCTGGTTGGTGACCATGGCGCACGATAATGAAGCTATGAGGGTTCTGAGGTCGACCAGGCTGAGGCCCGCTCATCTTGATGTTTTCTGGAACTTATGCCGTCCTATGGGAGCCAATCGAGGTTTCCTGTACAAGGGCGCTTAAACATCGCGCACGATATCGCCGCTCGAGCCCTCCGTTTAACTGCACTTGTTGAACTCACTGCAGGTCCGGACCGTTTGGAAGTGTCGCAACTGTGCTCGGGATAAGAACCTTCCGGACAACGGTGGTTGATGGCGCTTCATCTTGAGGGAATGGCCCCGAGCAGTATGTCTTTCGTCCGACGCCCTCCTGCCAAGTCCTATGGGGGCCGGGCGGCATAACCTCACCTATGAACTCACTCTGCTGTTTGCAGGTTAAGATTTCATCTAATGGCGCTGGATTGCACTGCAACATTCCCGGGCGGAGCCCTAGTGCAGCTCCGGGGAGGTAGCAGCTTTTTGGCAGTGCCCTCTATAGGTACAAAGGTTTAAGCCCAGCGACATTCGCTTACCGGCGGTTTGTGGGCTGCATCCTGAGGGGCTATTGGCACTTCCCCCTTTTCCCCGGGCAATCATCTGGAGTCCGCTAATCTTTTGTGGGGAGGGACTTTTGTACACCCGTGACTTGTCAACATCTGTTTTTCTAGTGATTTTTTTCCCCCCTGAGGCGCTGCCGCTGTGCGGCCGCCTGCCACAGGGTTGCTCCACCCTACAACCCCTGTTAGTTGACATTTGGGTGTTGCCGCCACCCTCTGAGGGGTCTCAGAGTTGATGCTGCGGCCTGCTCTCCAGTCCTTGAGCCCGTTCAACCGTCCGGCCCTGCTTAGGCCGAAGGCCCCGTGTGTAAGCCACCGACACAACGACCTCGCGCGTACCCGCCGCCGTCCTCTACACTTATCCGGATGGGCGAAGGTGTAATGCGGGGTCACTGTTTGAGTCAGACTGTGATTGGCTGGTCAATTGCGTCGAATCCCGGCCATCGTCCCGGAGGTGGCCTTTGCCATGCTTTTTAGCCAACGTAACCGAGTCGTTCCACCCAACTGAGTTTCATTTATGCGTGACGGTCTTGCCGCATATACTCTGAACCCCTCGGCTATTATCCATGCGGTTGGCTCGATTATAAGGGTTGGAGCAGAACCGCGAAGAGGCTTGAGGCAGCATACGCGAGAGACCTGCTCCCCCGCGTGGCACCGCGCTTACCCACTTCTCGGCTCAGGTTATATACGAGTTCCCGTTAAGCCCAGCTTTTGATCGCTTGGGAGCGTAACCATCGCCCGGGGATGAGCTTTAGCTTGGACCGACCCTCGCCGCTACCTGGTTCGAGGCCAACAAGCAGCACAACCGGCCCTTACAATAACTGAAAGATACAGCCCGTACAGCACGCTAGCACTAGAGATGACGCTGCTACGGAGGTGGGCCGGGCTTGTGCCGGTTTGTACAGTAGCCCTGGGGTTGTGCACTACCAAATTTCCGTCGGGGTCCAGGTTTCAGGGAAAGTCGCGGTCTATACAGCAGGGGGACTGTGATGTTGTGGTCGCGCTCAGCTGATCGAT
+
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After removing them, I ran VICUNA and the process could end normally, apparently those issues doesn't bother MIRA. I have no contigs generated but it may be due to the PacBio encoding quality for the insert consensus which lower quality. I will investigate this point.