Snakemake - Missing MissingInputException in line 20: Missing input files for rule stringt:

Question

I am unsuccessful in running this small snakemake program for StringTie; the dry-run gives an error of MissingInputException for the "rule stringt", I am unable to understand the issue here since the same directory structures works fine for a different snakemake program.

I have already generated the bam files using "hisat2" and is located in the directory : "/alternate_splice/bam_out/" which is stored as "bamdir".

The snakemake should be able to locate the input file since the names and location are appropriate, however it throws an error every time.

I did look at the pervious snakemake related questions, however could not solve this issue. If anyone can help me out here, it would be great!

There are 4 samples: for the wildcards, it takes the list from the directory which has the fastq files

~/alternate_splice/expdata$ ls -ltr
 -rw-r--r-- 1 shivani domain^users 1306438351 Jan  2 09:46 TL-19-DA4299_T_RSQ1_2.fastq.gz
 -rw-r--r-- 1 shivani domain^users 1185743896 Jan  2 09:46 TL-19-DA4299_T_RSQ1_1.fastq.gz
> 
 -rw-r--r-- 1 shivani domain^users 1896352262 Jan  9 08:49 TL-20-24D57D_T_RSQ1_2.fastq.gz
 -rw-r--r-- 1 shivani domain^users 1730191383 Jan  9 08:49 TL-20-24D57D_T_RSQ1_1.fastq.gz
> 
 -rwxr-xr-x 1 shivani domain^users 3215901253 Mar 25 10:28 BREAST_817_N1_RNA_REP1_1.fastq.gz
 -rwxr-xr-x 1 shivani domain^users 3396212102 Mar 25 10:36 BREAST_817_N1_RNA_REP1_2.fastq.gz
> 
 -rwxr-xr-x 1 shivani domain^users 3633768287 Mar 25 10:45 BREAST_792_N1_RNA_REP1_1.fastq.gz
 -rwxr-xr-x 1 shivani domain^users 3932340643 Mar 25 10:54 BREAST_792_N1_RNA_REP1_2.fastq.gz

The snakefile here: "bamdir" = directory to bam output "geneGTF" = locating GFT file

Two rule: 1. stringt 2. merge

 (SAMPLE,)=glob_wildcards("/home/shivani/alternate_splice/expdata/{sample}_1.fastq.gz")
#(SAMPLE,)=glob_wildcards("/home/shivani/alternate_splice/bam_out/{sample}.bam")
 bamdir = "/home/shivani/alternate_splice/bam_out/"
 refere_genome = "/home/shivani/ccb1_shivani/hisat2_trans_bam/hisat2_index/"
 geneGTF = "/home/shivani/stringtie_run/Homo_sapiens.GRCh37.87.gtf"

rule all:
        input:
               expand(bamdir+"{sample}_transcript.gft",sample=SAMPLE),
               expand(bamdir+"{sample}_abundance.tsv",sample=SAMPLE),
               expand(bamdir+"{sample}_coverage.gtf",sample=SAMPLE)
               expand(bamdir+"{sample}_stringtie_merge.gtf",sample=SAMPLE)


rule stringt:
        input:
                bm=bamdir+"{sample}.bam",
                gtf=geneGTF,
                tname="{sample}"
        output:
                tscripts=bamdir+"{sample}_transcript.gft",
                abund=bamdir+"{sample}_abundance.tsv",
                cov=bamdir+"{sample}_coverage.gtf"
        shell:
                """stringtie -p 4 -e -c 3.5 -G {input.gtf} -o {output.tscripts} -A {output.abund} -C {output.cov} -l {sample}{input.bm}"""


rule merge:
       input:
               trnsgtf=bamdir+"{sample}_transcript.gft",
               ggtf=geneGTF
       output :bamdir+"{sample}_stringtie_merge.gtf"
       shell:"stringtie -p 4 --merge -G {input.ggtf} -o {output} {input.trnsgtf}"

The dry run for this snakemake: instead of using "Snakefile" as designated name, I have used "stringtie_trans"

snakemake -n -r -s stringtie_trans

The output is as follows:

MissingInputException in line 20 of /home/shivani/alternate_splice/stringtie_trans ::::
Missing input files :::: for rule stringt: BREAST_792_N1_RNA_REP1

Unrelated to the error you report... -l {sample}{input.bm} I think should be -l {wildcards.sample} {input.bm} (note the space) — dariober
Yes, I removed the "tname = {sample}" part and it worked well! However when using :: -l {wildcards.sample} {input.bm} it does not understand and shows an error ::: Wildcard not defined for 'sample' — SdkVish

Maarten-vd-Sande Maarten-vd-Sande · Accepted Answer · 2020-04-02T08:14:00

As the input you have a variable called tname which isn't used, and I think it is there by mistake:

rule stringt:
        input:
            bm=bamdir+"{sample}.bam",
            gtf=geneGTF,
            tname="{sample}"  <-- should this be here?

Snakemake - Missing MissingInputException in line 20: Missing input files for rule stringt:

1 Answers