B-234 The Quality in NGS: Do You Know How Much We Lost?

Abstract

Abstract Background Next generation sequencing (NGS) is a new high throughput approach for genomic sequencing and variations detection. The consumption of NGS is numerous, such as times, money and human resource, therefore, some hospitals provided the service but delivered the specimen to other laboratories. Hence, we aimed to establish a procedure to monitor the quality and integrity of report. Method Here we used one clinical case to elucidate the whole process of raw sequence data analysis by free resource, a website usegalaxy.org and the IGV visualization software, with flowchart and quality control (QC) items. A delivered case will be an example to show how to monitor the QC. Results The flow chart showed the whole steps of analysis of NGS, including evaluation genetic variants in the report from individual laboratory. We processed the FASTQ file and analyzed the quality with three parameters. An overall picture of bases showed the phred score (QV) less than 28. Distribution of sequence lengths over all sequences highlighted a prominent peak at 110–119 bp. The mean QV of reads was around 26 with low quality (QV less than 20) in some reads. We then filtered the FASTQ file and the reads with length less than 45 bp or greater than 150 bp and QV less than 20 were filtered out. The data of the BAM file mapped to the designed panel were output. There were 8252 genome regions designed over 23 chromosomes (chr). A total number of reads was 1 085 653 with the average 131.6 reads (range: 0–31034; quartiles, IQ25: 1; IQ50: 27; IQ75: 152). 7746 of designed genome regions (93.9%) had reads less than 500 (quartiles, IQ25: 0; IQ50: 20; IQ75: 118). We further used IGV to check one of mutations which showed KRAS G12V in the report. KRAS G12V (chr.12: 25245350) was read 25 times with the variant allele frequency (C to A) of 24%. Conclusion We showed how to monitor the NGS quality by free resource. It is important to ensure high-quality data after we received the individual laboratory reports. That may help increase the quality of patient care and clinical service.