Error Running Manifest File

Technical Support
1

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
2

Hello @rylee650,

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

3

manifest-file.tsv (19.9 KB)

yes here it is!

4

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.

5

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

Screenshot 2025-04-04 at 2.13.27 PM
Screenshot 2025-04-04 at 2.13.27 PM1875×131 16.5 KB

6

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

8

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.

11

changing to .csv for phred64 helped!