Error Running Manifest File

version: qiime 2024.5

I have been struggling with this error for days:

Matplotlib created a temporary cache directory at /tmp/matplotlib-ffb4e81f because the default path (/home/qiime2/matplotlib) is not a writable directory; it is highly recommended to set the MPLCONFIGDIR environment variable to a writable directory, in particular to speed up the import of Matplotlib and to better support multiprocessing.
There was a problem importing /scratch/rylee650/test_manifest.tsv:

/scratch/rylee650/test_manifest.tsv is not a(n) PairedEndFastqManifestPhred64 file:

Found header on line 1 with the following labels: ['sample-id\tabsolute-filepath\tirection'], expected: ['sample-id', 'absolute-filepath', 'direction']

my import code:
module load qiime2/2024.5

qiime tools import
--type 'SampleData[PairedEndSequencesWithQuality]'
--input-path /scratch/rylee650/manifest-file.tsv
--output-path demux-paired-end.qza
--input-format PairedEndFastqManifestPhred64

the first few lines of my manifest file using cat -A manifest-file.tsv:

sample-id^Iabsolute-filepath^Idirection$
NX.VH01519_157.001.FLD_ill_097_i7---FLD_ill_0097_i5.V3-RG3-1^I/scratch/rylee650/NX.VH01519_157.001.FLD_ill_097_i7---FLD_ill_0097_i5.V3-RG3-1_R1.fastq.gz^Iforward$

NX.VH01519_157.001.FLD_ill_097_i7---FLD_ill_0097_i5.V3-RG3-1^I/scratch/rylee650/NX.VH01519_157.001.FLD_ill_097_i7---FLD_ill_0097_i5.V3-RG3-1_R2.fastq.gz^Ireverse$

to make the manifest code I used:

echo -e "sample-id\tforward-absolute-filepath\treverse-absolute-filepath" > manifest-file.tsv
for f in *_R1.fastq.gz; do
    sample=$(basename "$f" _R1.fastq.gz)
    echo -e "$sample\t/scratch/rylee650/${sample}_R1.fastq.gz\t/scratch/rylee650/${sample}_R2.fastq.gz" >> manifest-file.tsv
done

and my samples are for sure phred64:
IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII9IIIIIIIIIIIIIIIIIIIIIIIIIIII9II

ive tried so many ways to fix this and it doesn't seem to be working, maybe I am doing it wrong. ive tried rewriting the header using vim as well
my file is ascii and it was edited on a Mac.

please help!!!

1 Like

Hello @rylee650,

Can you attach the manifest file that isn't importing successfully?

manifest-file.tsv (19.9 KB)

yes here it is!

Hello @rylee650,

Are you sure this file is the one that gave you the error you attached? The bash loop you provided uses the sample-id/forward-path/reverse-path format, while this uses the sample-id/path/direction format. Also, the file names are different.

Also, the error states:

['sample-id\tabsolute-filepath\tirection']

which looks like a) the "d" in "direction" was missing and b) the tabs were not interpreted as tabs, but rather as literal "\t"s.

also not sure why that error is like that. this is my most recent error:

yes that's true, I remade the manifest file a few times so that was an earlier code, this is what I used:

echo -e "sample-id\tabsolute-filepath\tdirection" > manifest-file.tsv

for f in *_R1.fastq.gz; do
    sample=$(basename "$f" "_R1.fastq.gz")  # FIXED the quote issue
    echo -e "$sample\t/scratch/rylee650/${sample}_R1.fastq.gz\tforward" >> manifest-file.tsv
    echo -e "$sample\t/scratch/rylee650/${sample}_R2.fastq.gz\treverse" >> manifest-file.tsv
done

however I have also used this code to remove white spaces: sed -i 's/ */\t/g' manifest-file.tsv

Hello @rylee650,

The problem is that the PairedEndFastqManifestPhred64 format actually expects comma-delimited values, not tab-delimited ones.

There are two solutions: 1) change the delimiter using this format, or 2) use PairedEndFastqManifestPhred64V2 as the input format, and revert back to the sampleid-/forward-absolute-path/reverse-absolute-path layout.

Sorry for the confusion.

changing to .csv for phred64 helped!