Weird Illumina MiSeq question, some help with import?

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Long time QIIME1 user, new QIIIME2, trying to understand how to import files. We have 3 files with multiple samples from the Illumina MiSeq sequencer. The Files are R1 - forward, R2 -barcodes, and R3 reverse. Multiple samples are included in these files.

31%20PM

head of forward:

@MISEQ01:230:000000000-BJB48:1:1101:15468:1595 1:N:0:
TACGGAGGATGCGAGCGTTATCCGGATTTATTGGGTTTAAAGGGTGCGCAGGCGGTCTGTTAAGTCAGCGGTCAAAGCCCGGGGCTCAACCCCGGCCCGCCGTTGAAACTGGCAGTCTCGAGTTGGAGAGAAGTATGCGGAATGCGCGGTG
+

1>111111B1B110A0AE0FEG000AEG1FG11/AFE121A1/B/A//A//>//>/>0FF221BB21@//?//111BBF//<@BCEC1GGCC//BCC?@C?CC..DBGD0/</.<CD0/0.GE0/0//..CC:00CC.??@E0B.C-?E?
@MISEQ01:230:000000000-BJB48:1:1101:15537:1602 1:N:0:
CCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCC

AABCCCCCCCCCGGGGGGGGGGGGGGGGGGGGGGGGFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFADFFFFFFAC
@MISEQ01:230:000000000-BJB48:1:1101:18142:1620 1:N:0:
TACGTATGGTGCAAGCGTTATCCGGATTTACTGGGTGTAAAGGGAGCGCAGGCGGTGCGGCAAGTCTGATGTGAAAGCCCGGGGCTCAACCCCGGGACTGCATTGGAAACTACCGAACTAGAGTGTCGGAGAGGTAAGTGGAATTCCTAGT

I have been trying to import these files into QIIME2 and have been using qiime2 import using:

qiime tools import --type EMPPairedEndSequences --input-path ~/QIIME2_2_fastq --output-path ~/QIIME2_2_fastq/emp-paired-end-sequences.qza

qiime tools import --type 'SampleData[PairedEndSequencesWithQuality]' --input-path ~/QIIME2_2_fastq --source-format CasavaOneEightSingleLanePerSampleDirFmt --output-path ~/QIIME2_2_fastq/emp-paired-end-sequences.qza

Neither one works, both error respectively with:

Missing one or more files for EMPPairedEndDirFmt: 'barcodes.fastq.gz'

Missing one or more files for CasavaOneEightSingleLanePerSampleDirFmt: '.+_.+_L[0-9][0-9][0-9]_R[12]_001\.fastq\.gz'

I'm just wondering what the issue is? Should I demultiplex the samples in each of the runs? Should I create a manifest? Thank you.

2

Hi @ben,

The issue is simply that the file names are incorrect for EMP format, even though the format is correct. Try this:

  1. Make a new directory raw-seqs
  2. run the following commands (you will probably need to change the filepaths):
cp lane1_NoIndex_R1_001.fastq raw-seqs/forward.fastq.gz
cp lane1_NoIndex_R1_002.fastq raw-seqs/barcodes.fastq.gz
cp lane1_NoIndex_R1_003.fastq raw-seqs/reverse.fastq.gz
  1. Try importing again as EMP format

Let me know if that helps!

2 Likes
3

Great, works like a charm - it’s kind of funny because I tried this before and I think it had something to do with the way the files are formatted. I think that it was this issue, some formatting I did and voila, it works. Thank you again.

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