16.2.22 Illumina run setup: FAQ

We are considering using the Institute’s RNA sequencing services. With whom should we consult?

Sequencing is performed at the G-INCPM. They have two machines: NextSeq and NovaSeq (the HiSeq machine is no longer in service). Contact their Genomics unit headed by Hadas Keren-Shaul) regarding protocols, machines and prices.
- Usually, one gives the lab total RNA, and they prepare the libraries.
- We recommend at least three biological replicates for each treatment or control, as well as 15 million reads per sample and single reads for RNA-seq at the gene level (no isoforms analysis).
- If you run the samples in NovaSeq, they must have double unique indices.
Weizmann scientists can use the services of the Sandbox advanced genomics technological laboratory which include: Mars-seq (preparation of libraries for RNA-seq), Illumina Nextseq sequencing, 10x genomics (single cell RNA-seq) and PacBio (long read sequencing).
Bioinformatics analysis can be performed by LSCF bioinformatics unit staff or by the bioinformatics unit at the G-INCPM. A kickoff meeting between your lab and the chosen bioinformatics unit before starting the experiment is strongly recommended.

What do I need to prepare before setting up an NGS Sandbox run?

You should have an account (userID and password) on the SusanC3 server. To obtain one, contact the bioinformatics team at biopipe@weizmann.ac.il.
Right after a sequencing run starts, you should log in to the NGS Pipeline (SusanC3) website and register the run.
If you request demultiplexing and quality control services, you are required to upload a sample sheet.
The sample sheet must be prepared in a format (csv or xlsx) corresponding to one of the following three experiment protocols: Illumina-compatible indexing (TruSeq RNA-Seq and others), Mars-Seq or 10x Genomics (single cell RNA-Seq).
We recommend that you test the sample sheet format here
For more details see: https://bbcunit.atlassian.net/wiki/spaces/BP/pages/2543551200

Where are my fastq files located?

In the SampleSheet file, for each sample, you must specify the userID of the user who will receive the sequences. Different samples can be “demultiplexed” - i.e. delivered to different users.
The fastq files associated with each sample are placed in the specified user’s folder on stefan. For example, if in the SampleSheet, the samples Orel_tar_twist and Miri_scMARS belong to yaaraf, the output will be in ya/yaaref (http://stefan.weizmann.ac.il/users/ya/yaaraf/200520_A00929_0070_AHL775DRXX/)

How do I download my fastq files from a sequencing run?

You can access and download the sequencing data via WGET on UNIX (see detailed command below), or from your web browser.

Should we consider using microarrays or alternatives to RNA-Seq protocols at the genomics and sandbox unit?

If you prefer, you can prepare the libraries by yourself, and them bring them to run in the G-INCPM. There is a kit from Lexogen, with a dual indexing module.
Using microarrays might be an acceptable way to go, but the technology is old and results might be more difficult to publish. The Affymetrix representative is Amos Grundwag. If you buy a kit with reagents for hybridization, the lab work at WIS will cost ~300 shekel per sample. You will still need bioinformatics analyses as provided by our unit.