Snakemake pipeline not attempting to produce output? - snakemake

Question

I have a relatively simple snakemake pipeline but when run I get all missing files for rule all:
refseq = 'refseq.fasta'
reads = ['_R1_001', '_R2_001']
def getsamples():
import glob
test = (glob.glob("*.fastq"))
print(test)
samples = []
for i in test:
samples.append(i.rsplit('_', 2)[0])
return(samples)
def getbarcodes():
with open('unique.barcodes.txt') as file:
lines = [line.rstrip() for line in file]
return(lines)
rule all:
input:
expand("grepped/{barcodes}{sample}_R1_001.plate.fastq", barcodes=getbarcodes(), sample=getsamples()),
expand("grepped/{barcodes}{sample}_R2_001.plate.fastq", barcodes=getbarcodes(), sample=getsamples())
wildcard_constraints:
barcodes="[a-z-A-Z]+$"
rule fastq_grep:
input:
R1 = "{sample}_R1_001.fastq",
R2 = "{sample}_R2_001.fastq"
output:
out1 = "grepped/{barcodes}{sample}_R1_001.plate.fastq",
out2 = "grepped/{barcodes}{sample}_R2_001.plate.fastq"
wildcard_constraints:
barcodes="[a-z-A-Z]+$"
shell:
"fastq-grep -i '{wildcards.barcodes}' {input.R1} > {output.out1} && fastq-grep -i '{wildcards.barcodes}' {input.R2} > {output.out2}"
The output files that are listed by the terminal seem correct, so it seems it is seeing what I want to produce but the shell is not making anything at all.
I want to produce a list of files that have grepped the list of barcodes I have in a file. But I get "Missing input files for rule all:"

Answer 1

There are two issues:
You have an impossible wildcard_constraints defined for {barcode}
Your two wildcards {barcode} and {sample} are competing with each other.
Remove the wildcard_constraints from your two rules and add the following lines to the top of your Snakefile:
wildcard_constraints:
barcodes="[A-Z]+",
sample="Well.*",
The constraint for {barcodes} now only matches capital letters. Before it also included end-of-line matching (trailing $) which was impossible to match for this wildcard as you had additional text in the filepath following.
The constraint for {sample} ensures that the path of the filename starting with "Well..." is interpreted as the start of the {sample} wildcard. Else you'd get something unwanted like barcode=ACGGTW instead of barcode=ACGGT.
A note of advice:
I usually find it easier to seperate wildcards into directory structures rather than having multiple wildcards in the same filename. In you case that would mean having a structure like
grepped/{barcode}/{sample}_R1_001.plate.fastq.
Full suggested Snakefile (formatted using snakefmt)
wildcard_constraints:
barcodes="[A-Z]+",
sample="Well.*",
refseq = "refseq.fasta"
reads = ["_R1_001", "_R2_001"]
def getsamples():
import glob
test = glob.glob("*.fastq")
print(test)
samples = []
for i in test:
samples.append(i.rsplit("_", 2)[0])
return samples
def getbarcodes():
with open("unique.barcodes.txt") as file:
lines = [line.rstrip() for line in file]
return lines
rule all:
input:
expand(
"grepped/{barcodes}{sample}_R1_001.plate.fastq",
barcodes=getbarcodes(),
sample=getsamples(),
),
expand(
"grepped/{barcodes}{sample}_R2_001.plate.fastq",
barcodes=getbarcodes(),
sample=getsamples(),
),
rule fastq_grep:
input:
R1="{sample}_R1_001.fastq",
R2="{sample}_R2_001.fastq",
output:
out1="grepped/{barcodes}{sample}_R1_001.plate.fastq",
out2="grepped/{barcodes}{sample}_R2_001.plate.fastq",
shell:
"fastq-grep -i '{wildcards.barcodes}' {input.R1} > {output.out1} && fastq-grep -i '{wildcards.barcodes}' {input.R2} > {output.out2}"

Answer 2

In addition to #euronion's answer (+1), I prefer to constrain wildcards to match only and exactly the list of values you expect. This means disabling the regex matching altogether. In your case, I would do something like:
wildcard_constraints:
barcodes='|'.join([re.escape(x) for x in getbarcodes()]),
sample='|'.join([re.escape(x) for x in getsamples()]),
now {barcodes} is allowed to match only the values in getbarcodes(), whatever they are, and the same for {sample}. In my opinion this is better than anticipating what combination of regex a wildcard can take.