|
| Status |
Public on Jan 30, 2021 |
| Title |
Sensitivity_Testing_MIPP-seq2 |
| Sample type |
SRA |
| |
|
| Source name |
50ng Sensivitiy Testing Part 2
|
| Organism |
Homo sapiens |
| Characteristics |
tissue: blood and cell line derived genomic DNA
experiment: Dilution series
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
MIPP-Seq: Unique primers are designed to prevent sharing binding sites and avoiding known mutations in the individual and general population but are within 250 nucleotides of the target mutation. Once designed, unique 10nt barcodes are appended to the reverse primer, along with the standard Ion Torrent platform specific adapters. The primers designed above amplify targets using reduced cycling (20cycles) with a high-fildelity enzyme and minimal amount of DNA (25-50ng). As all primers are unique and can be identified using the primer sequence, recations from up to 96 differnet mutations are pooled and PCR purified, before being diluted to 100pm for sequencing on the Ion Torrent S5 with 400nt reads platform for ultra-deep coverage.
|
| |
|
| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Ion Torrent S5 |
| |
|
| Description |
50ng Sensivitiy Testing Part 2
|
| Data processing |
Library strategy: MIPP-seq
See MIPP-seq publication
First, all BAMs were converted to fastq using bedtool’s bamtofastq tool. Next, the samples were demultiplexed using the unique 15nt barcodes (5nt of the primer and 10nt index) using FASTX toolkit’s fastx_barcode_splitter (--bol --mismatches 3) resulting in fastq files for each primer set.
Indel correction was performed using Pollux[44] (-n false -d false -h true -s false -f false), and barcode and quality trimming were performed using the cutadapt [45]tool (-u 10 -q 10).
aligned to the reference genome using default settings in BWA-mem with local indel realignment being performed with GATK 3.7 IndelRealigner[46] (-greedy 1200 -maxReads 2000000 -maxInMemory 1500000) with indels present in gnomAD being used as a reference. Primer binding sites were removed using the bamclipper tool[47] with default settings. Variants were called across the length of each amplicon using Samtools mPileup with the settings: q=20, Q=20.
Genome_build: hg19
Supplementary_files_format_and_content: text TDT files containing all primers sites, sequences and the allelic fractions of mutations and correseponding background error rates
|
| |
|
| Submission date |
Jan 29, 2021 |
| Last update date |
Jan 31, 2021 |
| Contact name |
Ryan N Doan |
| Organization name |
Boston Children's Hospital
|
| Street address |
3 Blackfan Circle, CLS 15040
|
| City |
Boston |
| State/province |
MA |
| ZIP/Postal code |
02115 |
| Country |
USA |
| |
|
| Platform ID |
GPL23934 |
| Series (1) |
| GSE165780 |
MIPP-Seq: Ultra-sensitive rapid detection and validation of low-frequency mosaic mutations |
|
| Relations |
| BioSample |
SAMN17676164 |
| SRA |
SRX9975364 |
