High throughput sequencing of multiplexed PCR amplicons with Short Tandem Repeats (STRs) requires software solutions that sort the information and allow a comprehensive overview of the results without overwhelming the data analyst with details. Here, we present an updated version (2.0) of the STR analysis tool STRinNGS. It is freely available as a Docker image or zip file ready for downloading. STRinNGS predicts genotypes using criteria for read depth, noise, flanking region lengths, mismatches in the flanking regions, locus balance, and heterozygote balance. Warning flags highlight suspicious genotypes as well as suspicious sequences that are not identified as either noise or alleles in the result table used for the manual analysis. STRinNGS analyses both the STR and the flanking regions, and names the alleles according to the STRidER guidelines as well as an in-house nomenclature that also include variants in the flanking regions. Furthermore, STRinNGS generates files with analysed data in a format that may be uploaded directly to the STRidER database. We re-analysed 627 sample files from eight different MiSeq FGx runs with STRinNGS v2.0. The samples were previously typed with the ForenSeq™ Signature Prep Kit and analysed with STRinNGS v1.0 and the Universal Analysis Software. Apart from three poorly performing loci with large heterozygote imbalances (Penta E and D22S1045) or frequent single nucleotide errors (DYS461), only 58 genotype calls (0.2 %) had to be manually corrected and only 14 genotype calls were discordant with the previous analyses. The discordant calls were primarily caused by manual oversights and in every case, the STRinNGS v2.0 analysis was correct.