It is recommended t o provide 3 micrograms of DNA with an OD 260/280 value of 1.8 and OD 260/230 value of 2.0, to get good quality results. Dissolve DNA in low TE(pH 8.0) or Nuclease Free Water.
Genome Sequencing provides a comprehensive view of coding, non-coding and mtDNA. It is reliable and sensitive in detecting all types of variants (SNVs, Indels and CNVs). Whereas , exome sequencing gives a targeted view of only protein-coding regions of the genome.
Twist – Human Comprehensive Exome and mtDNA Library preparation kit is being used.
To get good coverage of the target region 6GB of data to be generated.
Peripheral Blood or saliva or Buccal swab can be used.
Exome sequencing can detect SNPs, and Insertions/Deletions.
Fresh leaves are best for good results. The leaves are to be washed with distilled water to remove dust particles and cut into pieces. These pieces are to be snap frozen in liquid nitrogen and stored at -80o C for longer storage or transported in dry ice for processing.
Any DNA isolation procedure that retains high molecular weight molecules is good to obtain both Nuclear and Mitochondrial DNA.
There are two methods for targeted sequencing. 1. PCR based Enrichment and 2. Propbe base capture. In the first method, targeted regions are amplified in 1 or 2 multiplex PCR amplification, In the second method, the regions of interest are captured by hybridization to biotinylated probes and then isolated by magnetic pull down. Target enrichment captures 20 kb–62 Mb regions, depending on the experimental design. Since these are custom design a minimum sample number is required based on the design.
It depends on the gene size.
For germline variants, in case of PCR based enrichment method depth of 500X-1000X should be prefferred. If it is capture method, 50X depth should be achieved. For somatic variants, the depth should be increased for both methods.
A single method many not work for all kinds of experiments. You can try focused sonication (Covaris or Biorupture) to generate DNA fragments 0f size 200-500 bp. We recommend testing the ChIP DNA on a 2% gel or any Fragment analyzer (Bioanalyzer, TapeStation, Qiaxel etc.) before submitting the samples.
Organisms with annotated reference sequence are suitable.
Sequencing of all the hypervariable regions is available. Depending on the customer’s request, specific primers are used to target, amplify and sequence the regions from V1- V9.
It depends on the level of degradation. You can share the gel pictures along with ladder details to our support team to get the chance of success.
V3-V4 region is widely used. It may vary based on the sample source.
As per the published literature 6 biological replicates are required. However, majority of the studies show using 3 biological replicates prudential.
RNA Sequencing provides quantifiable results for each transcript. It can detect novel transcripts.
Ribosomal RNA (rRNA) constitutes more than 90% of total RNA. Performing RNA -Seq without Poly-A enrichment or rRNA depletion will generate the rRNA reads. Transcriptome that has been rRNA depleted is often considered as total RNA, including both mRNA and non-coding RNA (except miRNA).
5 ug of total RNA.
10 million reads.
No enrichment is required for microRNA sequencing because adapters are selectively ligated to mi RNAs only.
Any cell type that expresses polyadenylated mRNA molecules is compatible with single-cell RNA-seq work flow.
Single-Cell Gene Expression is compatible with fresh, fixed (methanol), and cryopreserved single-cell or single-nuclei suspensions. Organisms with well annotated reference genomes are suitable for SCGE.