# Nanopore Fastq not recognized by Mothur

**URL:** <https://forum.mothur.org/t/nanopore-fastq-not-recognized-by-mothur/22076>\
**Category:** Commands in mothur\
**Created:** [February 23, 2024, 2:47pm UTC](https://forum.mothur.org/t/nanopore-fastq-not-recognized-by-mothur/22076 "2024-02-23T14:47:14Z")\
**Posts on this page:** 6\
**Page:** 1

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**Author:** ![Alexandre\_Thibodeau](https://avatars.discourse-cdn.com/v4/letter/a/e480ec/32.png) [@Alexandre\_Thibodeau](https://forum.mothur.org/u/Alexandre_Thibodeau)\
**Post date:** [February 23, 2024, 2:47pm UTC](https://forum.mothur.org/t/nanopore-fastq-not-recognized-by-mothur/22076/1 "2024-02-23T14:47:14Z")

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Hello everyone! Got our first nanopore sequencing run done. I am exploring my possibilities for analysis and being a long time user of Mothur, I am trying to use Mothur for it.

I took the fastQ files that pased the Nanopore basecaling and that were generate from their program MinKNOW.

I concatenated all files per barcode to get a single fastq file per barcode then, I went to mothur.

My plan is to used fastq.info as a start t generate the fasta files. Unfortunately, mothur do not recognized the file as fastq.

"  
mothur \> fastq.info(file=nanopore\_test1.file)  
[WARNING]: Blank fasta name, ignoring read.  
[WARNING]: missing sequence for , ignoring.[WARNING]: expected a name with + as a leading character, ignoring.[WARNING]: missing quality for , ignoring.[WARNING]: Blank fasta name, ignoring read.  
"

This is an exemple of what the file look like.(Copy paste)

@38676c8c-7575-4957-a161-5ce1fa18506a runid=2ecfd430e213202cb6f7538082e8ad79ba5f9760 read=16 ch=390 start\_time=2024-01-31T16:44:23.549452+00:00 flow\_cell\_id=FAX64935 protocol\_group\_id=16s\_essai1 sample\_id=no\_sample barcode=barcode96 barcode\_alias=barcode96 parent\_read\_id=38676c8c-7575-4957-a161-5ce1fa18506a basecall\_model\_version\_id=dna\_r10.4.1\_e8.2\_400bps\_fast@v4.2.0  
TGTTATGTCCTATTTACTTCGTTCAGTTACGTATTGCTGGTGCTGCTGAACGGTCATAAGAGTCCACCATTTAACCTTTCTGTTGGTGCTGAATATTGCAGAGTTTGATTATGGCTCAGGATGAACGCTGGCGGCGTGCCTTAATCATGCCAAGTCGAGCGAACGGACAGAAGCTTGCTCTTCTGATGTTAGCGGCGGACGGGTGAGTAACACGTGGATAACCTACCTATAAGACTGGGATAACTCGGGAACCGGAGCTAATACCGGATAGTATTTTGAACCGCATGGTTCAAAATATTATCGGTATTGGGTTCCGAAGTTATGCCGGTCTTATAGGTAGGTTATCACGTCTACTGGCCCGTTCCGCCGCTAACGTCCGGAGGGCTCCTCGTCCATTTCGCTCGACTTGCAGTGTATTAGGCACACCGCAGCGCTGATCCTGAGCCATGAAACTCTGCCGATATCAGCACCGACGGAAAGGTTAAATGATCAACTCTATAACGTTCGCGGCACCACTGGATA  
+  
$$‘(&((+%%%$&$((,.)**\>9+)_310068311887568+_+'(39\<\<\<\<=\<\<\<1,)22\>4==@CDM=E???FCB;7;4427;A@@432200.+1003.731589DBA89=C\<989EMBABA\>\>?\<\<\>A@C777\>;96&6912@\<=52=:\<:\<;?;33\>\>876:&(-9/,+/+34/30.85:\<\>::7\>@\>==B?@8687;00094599922254A,/34034C872(_-))%%7771101&/2=;\<67:=@545–.234599;7&&%-,./.,9677822-/00/))_/**)_/1-,14.–$#&(-%%&&,)%&%&%(_(%%(’+()$$,/‘%)(’‘**&&%)(_$$)##%.0-+)_-(()0,/$$$&(#$&‘’‘_‘$$#$&$%$%,’&'33223)()_,3)’$&$‘&####%)),+%&&,’%,++‘_%#&'_,)’‘)0&&%%.+_.‘’##&‘’()_()($$%%)())))0+_0,_%$’((%&$$&)_&&#‘$$%&$$’$&&(())_&##%‘)_%  
 @c0f18f2d-ca5d-48e6-9091-3abb99f64e94 runid=2ecfd430e213202cb6f7538082e8ad79ba5f9760 read=26 ch=443 start\_time=2024-01-31T16:44:24.549452+00:00 flow\_cell\_id=FAX64935 protocol\_group\_id=16s\_essai1 sample\_id=no\_sample barcode=barcode96 barcode\_alias=barcode96 parent\_read\_id=c0f18f2d-ca5d-48e6-9091-3abb99f64e94 basecall\_model\_version\_id=dna\_r10.4.1\_e8.2\_400bps\_fast@v4.2.0  
ATGTAACCTACTGGTTCAGTTGCGTATTGCTGGTGCTGCTGAACGGTGATCAAGTCCACCATTAACCTACTTGCCTGTGGCTACTATCTTCTACGGCTACTTGTTACAACTTCACCCCAATCATTTACCACCTTCGACGGCTAGCTCCTAAAAGGTTACTCCACCGGCTTGGGTGTTACAAACTCTCGTGGTGACGGGCGGTGTGTACAACCGGGAACATATCACCAGTAGCATGCTGATCTACGATTACTAGCGATTCCAGCTTCAGTGTCCGTCGAGTAGACTACAATCCCGATTTAAACAACTTTATGGAATTGCTTGACCTCGCGGTTTCGCTGCCCTTTGTATTGTACACACCGCCCGTCACACCACGAGAGTTTGTAACACCCGAAGCCGTGGAGTAACCTTGCCAGAGCTAGCCGTAGAAGGTGGGACGAATCGTTGGGGTGAAGTCGTAACAAGGTAGCCGTAGAAGATCGAGCGACAGGCAGTTAGGTTGATCGGTGGACTCTGACCGTTCAGCAGCACCAGCAATACGTGGCT  
+  
%‘&)&)(()(’($&‘))(-’+('(-.779:79\>B?\<\<A9827;41/1&&&+((())735622334;8@92289KCHEB879:,-477:98\<;?..;2,&($#%+_’%%-4–,:;+(_‘##$(%%(334=/—/669B9889A??..\<46;7&’…&\<\>B\<211\<6&&\<=776;;9\>=98333230//201)(04%%%4965$$%$&,+,//-+‘’‘(’))+(&(()+0\>?=\>22265470346/./(_…-.22989891(()&‘((1%%###+)()3±+.0211’(($$)+)&%&+832–./799;;:1228=256::BB97:BBEAAD?\<@@AEBB:::\<334=\>?\<9:;=\<45520’().65576:8=?..D76@:68-'++0%&(-.01-(**’+‘459…/786’‘(0,\>\<–557\*()_%#%%+,-,&%$%%$+.5:@=::@:7699:80(&63-0-\*\*,8(&&-±…,+))044(&&&$&'%&##$)&&%&_+1/(/0886////24001(’‘_%%"#  
 @0a960ff5-f4a9-4850-9890-f8c52b8e70da runid=2ecfd430e213202cb6f7538082e8ad79ba5f9760 read=18 ch=409 start\_time=2024-01-31T16:44:24.549452+00:00 flow\_cell\_id=FAX64935 protocol\_group\_id=16s\_essai1 sample\_id=no\_sample barcode=barcode96 barcode\_alias=barcode96 parent\_read\_id=0a960ff5-f4a9-4850-9890-f8c52b8e70da basecall\_model\_version\_id=dna\_r10.4.1\_e8.2\_400bps\_fast@v4.2.0  
GTTGTGTAACCTACTTGGTTCGTTGCGTATTGCTGGTGCTGCTGAACGGTCATCAAGTCCACCATTTAACCTTTCTGTTGGTGCTGATATTGCAGAGTTTGATTCTGGCTAGGATGAACGCTGGCGGCGTGCCTAATACAGTAAGTCGAGCAAACGACGAGAAGCTTGCTCTCTGATGTAGCGGCGGACGGGTGAGTAACACGTGGATAACCTTGCCTATAAGCTGGGATAACTTGGGAAACCGGAGCTAATACCGGATAATATTTTGAACCGCATCATTAGAAAAGTAAAGACGGTCTTGCTGTTCACTTATTCAGTCGATGCGCGCTGCGTTAGGCTTAGTTGGTTCGGCCATCCATCGCTTACTAAGGCAACGGAGTGCGACGCCGACCTGAGAGGGTGATCGGCCACACTGGAACTGGAGCACGGTCCAGACTCCTACGGGAGGCAGCAGTGGGAATCTTCCGCAATCGGCAGAAAGCCTGACGGACAACGCCGCGTGAGTGATGGTCTTCGATCGTGATTTTGTTCGAGAGAACAAGAACATAGTAACTGAACGTCCCTGACGGTATCTAACCAGAAAGCCACCGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGATTATTGGGCGTAAAGCGACGCAGGCGGTTTTCTTAAGTCTGATGTGAAAGCCCCGGCTGAACCGGGGAGGGTACATGGAAACTGGAAGACTCGGTGCAGAAGAATTCCCTACTGCTGCCTCCGTAGGAGTCTGGACCGTGTCAGTTCCGTGTGGCCGTCAGCCTCAGGTCGGCTGTGGTCGGTTCCTTGAGTAAGCCGTTACCTTTACCGACTGGCTGATGCAGCGCGGTGCCGTCTATAAGACATTTTGAACCATCGTCAAGTGTATACCATTGTTGCTCCGGTTTTCCGGAAGTTTGCCGTCTTATGGTAGGTTGTCCACGGTGTCTCACCGTACCGCCGCTGAGCATGCCGAGAAGCAACTTTCTGATCCGGTTCGCTGGACTTGCAGCTCTTGACAGCGCCGCCAGCGTTGGTGTAGAGCCAAGTGAACTTGCGATGTCAGCAGGCCAACGGGAGGGGGCAATGGTCGACTGTATAACCGTACGGCCGCGCCCGGCGGTGGTGG  
+  
&,.‘&’+$##&'&)_,(0…(%’/))_67;\<?;;:BB@@@\>\>\>95;;112+''%(((/,548:;\<;?8767786//8,,,5;963011+-)%)-456\<1/055:++779/35\<;\<CB==\>A@?@CB112/0(&&%%+1)((;\<22@=2/0&%&).+--1,))&1,8/./62'33137=:B;78F@;:;@:\<\>\*\*\*\>\>\<\<\<.'''')+//7((&\*)#%')780\*)'+-)+;?AC\<;767C?AB??5443496568\<--=101018.))\*(%&&4B325:@=98))\<;;=91+-,)%(()9''&$%''&))-)%$)\*++.4)\*+((\*)--'(\*+,)('%'(.,\*&$%&(+\*)2=,+.,,-2/+((\*%&&&(+&#$%(+13/0B:6\*\*+9;:?\>\>4,.2+3’)9101.‘+.37687\<AA:70+**&&)$,9:9535++\<82’‘’.')/0.–0124\>4120**&&±678211A=9=\<101’'-4572_&2\*((&$#$&&%((%$&_-_-/_+&)(+&%&$#%&‘%%$#’&‘’%%',2\<??ACA?9868:@A@@IB???\>:+&/3?0/-.&%%+9;;=\<\>114\>\<==\>;657211:9\>\>=89:5456+_,_)-((++(&&%&)75,_.255)(((**()0,++9\>F53\*&+,((%%‘1\*&(3::76=E:88D0\*,-1)_\<\>=2…-$$%_)9?FA\>\>503(’&#$$_)+333:;;850&'_;?\>8667CFDFBC12/2’'(3454949:=:3212\*%$&+(_'&22((_++**%&&15)‘’_-,+/2A%%###‘&’&&#$')$$$_((_.00&+%%&),_‘&’&%%)$##&$$$),(().0/-‘&%_&$%)‘(%&&’%$$$1,)/±’&"$&%$%)(&'&,$$%$###$$%$&+.+_./5+()’%-,&&&$&($$%,._)‘’/2159:)((_)$$%$‘&#%&&&),%(&&%%$()%&’(,&&%(_(&+/-.,%%%‘’&'($#$#$+_&/((),)%&%‘_%%$%$$%_)#$’%%‘&’)/-0.-.((+&$%($$$%‘+1%$’‘’)/-.-%%"&,/.)&%(%%&).‘&’+_(‘%&%&’&#$')_+%$$$%%&&-\*,1\*\*&%#$‘-’%,016-,-'#$&$$$$

should I used another program to convert the file into fasta?

My general plan for the analysis is the following, feel free to comment on it.

fastq.info (to get fasta files)  
make.group (to create count file)  
merge.files (to get 1 single fasta file)  
unique.seqs (to get unique sequences and start the analysis)  
…  
and so on (when I guet this working I will toy with different parameters and see how our mock community and already MiSeq V4 sequences samples behave).

Kind regards,

---

<div class="post-metadata">

**Author:** ![Alexandre\_Thibodeau](https://avatars.discourse-cdn.com/v4/letter/a/e480ec/32.png) [@Alexandre\_Thibodeau](https://forum.mothur.org/u/Alexandre_Thibodeau)\
**Post date:** [February 26, 2024, 1:30pm UTC](https://forum.mothur.org/t/nanopore-fastq-not-recognized-by-mothur/22076/2 "2024-02-26T13:30:45Z")

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Just a quick update: I ran fastqc on a fastq file and the analysis is working fine. So it does not appear to be the file.

Kind regards,

---

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**Author:** ![Alexandre\_Thibodeau](https://avatars.discourse-cdn.com/v4/letter/a/e480ec/32.png) [@Alexandre\_Thibodeau](https://forum.mothur.org/u/Alexandre_Thibodeau)\
**Post date:** [February 26, 2024, 2:42pm UTC](https://forum.mothur.org/t/nanopore-fastq-not-recognized-by-mothur/22076/3 "2024-02-26T14:42:36Z")

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Another udate: changed format, still did not work.  
My problem was with my file: did a format that was column 1: “group” and then column 2: “fastq”. Replaced by doing a seperate iteration of fastq.info per fastq file and the fasta are forming correctly.

Finger crossed for the rest of the pipeline. I will post in another thread if i encounter a problem with other function.

Kind regards,

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**Author:** ![pschloss](https://yyz2.discourse-cdn.com/flex036/user_avatar/forum.mothur.org/pschloss/32/4_2.png) [@pschloss](https://forum.mothur.org/u/pschloss)\
**Post date:** [February 27, 2024, 6:54pm UTC](https://forum.mothur.org/t/nanopore-fastq-not-recognized-by-mothur/22076/4 "2024-02-27T18:54:42Z")

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`fastq.info` is expecting a fastq file as input. What does `nanopore_test1.file` look like?

---

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**Author:** ![Alexandre\_Thibodeau](https://avatars.discourse-cdn.com/v4/letter/a/e480ec/32.png) [@Alexandre\_Thibodeau](https://forum.mothur.org/u/Alexandre_Thibodeau)\
**Post date:** [February 27, 2024, 7:25pm UTC](https://forum.mothur.org/t/nanopore-fastq-not-recognized-by-mothur/22076/5 "2024-02-27T19:25:36Z")

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Hello! Thank you for answering back.

file looks like that (tab delimited)

barcode73 barcode73.fastq  
barcode74 barcode74.fastq  
…  
barcode96 barcode96.fastq

I went around it by using a different iteration of fastq.info for each file. It did not took too long to write.

Still toying with my first dataset (out of the nanopore raw fastq). I am putting together the pipeline while I am working on basecalling on my analysis computer to increase quality of the fastq. From what I saw and what I read, you cannot really do the basecalling on the nannopore sequencer (we have a MK1C), you have to use the basecaller on your computer to run a “super accurate” version and also toy with parameters for bacrode triming options which are not offered when you basecall on the sequencer. So I am doing 2 things in parallel.

Positive control looks ok on Epi2Me (Nanopore analysis) but I like having control on my sequencing analysis so I am building something in that sense.

My gut feeling is telling me that species taxonomic inference won’t be great but that full 16S will resolves correctly a lot of genuses that were missclassified or unclassified and probably won’t change a thing to alpha or beta diversity analysis overall results.

I will posted my best options when I have something that works on my hands.

Thanks again for reading me.

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**Author:** ![system](https://yyz2.discourse-cdn.com/flex036/user_avatar/forum.mothur.org/system/32/2_2.png) [@system](https://forum.mothur.org/u/system)\
**Post date:** [March 8, 2024, 7:25pm UTC](https://forum.mothur.org/t/nanopore-fastq-not-recognized-by-mothur/22076/6 "2024-03-08T19:25:49Z")

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