Combining items from several files into one matrix using Python

You need to join the file to the path:

from os import path, listdir

pth = sys.argv[1]  # get full path
myfiles = listdir(pth) # get list of all files in that path/directory
for f1 in myfiles:
    with open(path.join(pth,f1)) as f: # join -> pth/f1. with also closes your file
        tpp = f1.split("_",1)[0].split(".")
        tp = ".".join(tpp[0:3]) # same as tp=tpp[0]+'.'+tpp[1]+'.'+tpp[2]
        for line in f:
            # continue your code ...

You can write your code a little to be more more concise and more efficient using slicing, unpacking str.format and not repeatedly splitting:

from os import path, listdir
import sys
from collections import defaultdict

snps = defaultdict(lambda: defaultdict(str))
pth = sys.argv[1]  # get full path
myfiles = listdir(pth)  # get list of all files in that path/directory

with open("Fer1_INDELs_clones_filtered.csv","w") as out: # file to write all filtered data to
    out.write("Fermentor\tTrajectory\tChromosome\tPosition\tMutation\tGene\tEffect\t1.20.1\t1.20.2\t1.20.3\t1.30.1\t1.30.2\t1.30.3\t1.40.1\t1.40.3\t1.50.1\t1.50.2\t1.50.3\t1.60.1\t1.60.2\t1.60.3\t1.90.1\t1.90.2\t1.90.3\t1.100.1\t1.100.2\t1.100.3\t1.130.1\t1.130.2\t1.130.3\t1.200.1\t1.200.2\t1.200.3\n")
    for f1 in myfiles:
        with open(path.join(pth, f1)) as f:  # join -> pth/f1
            tpp = f1.split("_", 1)[0].split(".")
            tp = ".".join(tpp[0:3])  # same as tp=tpp[0]+'.'+tpp[1]+'.'+tpp[2]
            for line in f:
                ls = line.split()
                if line.find("#") == -1 and len(ls) > 6: 
                    print(line)
                    # use unpacking and slicing
                    chrom, pos, ref, alt, freq, typ, gene = ls[:7]
                    if len(alt) == 1:
                        # use str.fromat
                        snps["{}_{}-{}_{}_{}_{}".format(pos,ref,alt,chrom,gene,typ)][tp] = freq
                    elif len(alt) > 1:
                        # use enumerate
                        for ind,k in enumerate(alt.split(",")):
                            snps["{}_{}_{}_{}_{}_{}".format(pos,ref,k,chrom,gene,typ)][tp] = freq.split(",")[ind]
    traj = 1
    tp_list = ['1.20.1', '1.20.2', '1.20.3', '1.30.1', '1.30.2', '1.30.3', '1.40.1', '1.40.2', '1.40.3', '1.50.1', '1.50.2',
               '1.50.3', '1.60.1', '1.60.2', '1.60.3', '1.90.1', '1.90.2', '1.90.3', '1.100.1', '1.100.2', '1.100.3',
               '1.130.1', '1.130.2', '1.130.3', '1.200.1', '1.200.2', '1.200.3']
    for pos in sorted(snps):
        # split once and again use unpacking and slicing 
        pos1, mut, chrom, gene, typ = pos.split("_")[:5]
        tp_string = ""
        for tp in tp_list:
            #print(tp)
            if snps[pos][tp]: # empty value will be False no need to check len
                tp_string += "\t{}".format(snps[pos][tp])
            else:
                tp_string += "\t0/0"

        out.write(("F1{}\t{}\t{}\t{}\t{}\t{}\t{}\n".format(traj,chrom,pos1,mut,gene,typ,tp_string)))
        traj += 1