filterAndTrim returns no reads with least restrictive parameters

[HaigBishop]

Hello!

First time running some 454 16S sequencing through DADA2. Zero reads from all 40 samples passthrough the filterAndTrim function with my optimal parameters. So, of course I adjusted the parameters until I go to the least restrictive parameters (see below) still with no reads making it through.

I tried reinstalling all packages and using R 4.4.1 and 4.4.0.

Quality scores are low, but that could just be a 454 thing? Besides I have tried not using filters on quality.

The raw files are available on SRA under the project ID PRJNA287742. Not my data.

Hopefully it's a stupid mistake, any ideas?

filter_summary <- filterAndTrim(
    fwd = forward_reads,          # Input forward reads file(s)
    filt = filtered_forward_reads, # Output filtered forward reads file(s)
    compress = TRUE,               # Compress output
    truncQ = 0,                    # No truncation based on quality score
    truncLen = 0,                  # No truncation based on length
    trimLeft = 0,                  # No trimming from the left
    trimRight = 0,                 # No trimming from the right
    maxLen = Inf,                  # No maximum length restriction
    minLen = 0,                    # No minimum length restriction
    maxN = Inf,                    # Allow any number of ambiguous base calls (Ns)
    minQ = 0,                      # No minimum quality threshold
    maxEE = Inf,                   # No filtering based on expected errors
    rm.phix = FALSE,               # Do not remove phiX sequences
    rm.lowcomplex = 0,             # Do not remove low complexity sequences
    orient.fwd = NULL,             # No orientation check
    matchIDs = FALSE,              # No ID matching between paired reads
    id.sep = "\\s",                # Default ID separator (space)
    id.field = NULL,               # Automatic ID field detection
    multithread = FALSE,           # No multithreading
    n = 1e+05,                     # Default chunk size
    OMP = TRUE,                    # Use OMP multithreading
    qualityType = "Auto",          # Auto-detect quality encoding
    verbose = TRUE                # No verbose output
)

End snippet of console output:

Read in 7196, output 0 (0%) filtered sequences.
The filter removed all reads: D:\metagenome_datasets\unprocessed_datasets\16S_40_Wiggles\fastqs\filtered\SRR2082758_filtered.fastq.gz not written.
Warning message:
In filterAndTrim(fwd = forward_reads, filt = filtered_forward_reads,  :
  No reads passed the filter. Please revisit your filtering parameters.

[benjjneb]

What is the output of plotQualityProfile on the original fastq files?

[HaigBishop]

@benjjneb They all look like this. Is this typical for 454 data?