| Whole exome sequencing with bioinformatics analysis. |
Whole-Exome sequencing libraries will be prepared using Truseq Exome library prep kit or Sure Select XT Human All Exon v5.Biotinylated oligonucleotide capture probes, that are designed for the human exons are used to enrich the region of interest (whole exome) by hybridization.
The workflow involves shearing of DNA, repairing ends, adenylation of 3’ ends, followed by adapter ligation. At each step the products will be purified. The adapter sequences will be added onto the ends of DNA fragments to generate paired-end libraries. The resulting adaptor-ligated libraries will be purified, qualified and hybridized with an exome specific biotinylated capture library. After hybridization, the targeted molecules are captured on streptavidin beads. The resulting enriched DNA libraries will be multiplexed by adding index tags by amplification, followed by purification. Indexed captured library DNA will be assessed to check the
quality and quantity of the captured libraries.
Finally, indexed captured library DNA will be sequenced on Illumina NextSeq-500 to generate 2X75 bp sequence reads at 100X sequencing depth(~6 Gb). The generated sequence data will be checked for necessary quality control. A minimum of 80% of the sequenced bases will be of Q30 value. Sequenced data will be processed to generate
FASTQ files and uploaded on the FTP server for download. |
Genomic DNA:
1000ng -3.5μg of high quality, high molecular weight, non- degraded DNA with A260/280 ratio of 1.8-2.0 in nuclease free H2O or 10 mMTris, pH 8.0. The concentration of gDNA should be @ 50 -100ng/μL. Wherever possible or necessary, a gel should be run to assess the state of degradation of the DNA sample.
Note:
The DNA will be analyzed by Nanodrop and/or Qubit (Thermofisher scientific). If the quality of DNA is insufficient, A repeat sample will be requested.
Whole Blood:
Adult: Collect 9 mL# of EDTA anticoagulated (purple top) peripheral blood in 9mL tube.
Children: Collect 4mL#of EDTA anticoagulated (purple top) peripheral blood in 4mL tube.
Keep Stored at 4°C till transported to the lab. |
| Transcriptome (RNA) Sequencing and bioinformatics analysis. |
Illumina TruSeq RNA Sample Preparation V2 protocol is used to the libraries for mRNA sequencing. The first step in the workflow involves purifying the poly-A containing mRNA molecules using oligo-dT attached magnetic beads. Following purification, the mRNA is fragmented into small pieces using divalent cations under elevated temperature. The cleaved RNA fragments are copied into first strand cDNA using reverse transcriptase and random primers. Second strand cDNA synthesis follows, using DNA Polymerase I and RNase H. The cDNA fragments then go through an end repair process, the addition of a single ‘A’ base, and then ligation of the adapters. The products are then purified and enriched with PCR to create the final cDNA library.
Prepared libraries will be sequenced on Illumina Nextseq, 2x75 bp PE Sequencing. Upto 80% of the sequenced bases will be of Q30 value. Sequenced data will be processed to generate FASTQ files and uploaded on the FTP server for download. |
Total RNA
5 μg (250 ng/μl) total RNA, measured with Qubit fluorometer.(Un-degraded total RNA with RNA integrity number (RIN) ≥8, with 2100 Bioanalyzer or Tape Station is required)
Note:
The RNA will be analyzed by Nanodrop and/or Qubit (Thermofisher scientific). If the quality of RNA is insufficient, A repeat sample will be requested. |
| Targeted Amplicon Sequencing |
Targeted Amplicon sequencing is a cost and labour effective approach used in the following circumstances;
- When mutation screening is required in a large gene with multiple exons
- Mutation screening in a limited number of genes resulting in same disorder/phenotype
- Mutation screening in a limited number of genes responsible for closely related disorders/phenotype.
Amplicons generated by custom designed primers will be subjected to sequencing with Illumina NextSeq-500 to generate 2X75 bp sequence reads. |
Whole Blood:
Adult: Collect 9 mL# of EDTA anticoagulated (purple top) peripheral blood in 9mL tube.
Children: Collect 4mL#of EDTA anticoagulated (purple top) peripheral blood in 4mL tube.
Keep Stored at 4°C till transported to the lab
Amplicons will be produced by long range PCRs, in the size range of 2000 – 10000 bp from high quality DNA with A260/280 ratio of 1.8-2.0 in nuclease free H2O or 10 mMTris, pH 8.0. The concentration of DNA should be @ 200-500ng/μL for the long range PCRs.
Amplicons thus generated should be electrophoresed in 1%
agarose gel to assess the quality and are subjected to clean up
procedure prior to sequencing. |
