Guidelines

Paperwork and procedures

Step 1 - Request and approve a quote

  • Download, complete and send to us (igf@imperial.ac.uk) a ‌. We’ll contact you with a quotation. Follow this link for a quick walk through example on how to complete the quote request form
  • If you requested data analysis, we will need you to provide additional information regarding the experimental design, metadata and sample grouping. For this quote, we assume that each sample is only used ones for data analysis, should any sample be needed more than once (e.g. control vs group1 and control vs group2) the cost for analysis will be adjusted by counting the control sample(s) twice. 
  • Once we receive a signed copy of the QUOTE we’ll open a project. 

STEP 2 - submit your samples

  • Download, complete and send to us the sample submission form (link to form pending, in the meantime, contact igf@imperial.ac.uk for a copy)
  • Prepare and deliver samples/libraries in accordance with our sample submission guidelines
  • Samples/libraries: Drop off to the facility in clearly labelled tubes, if more than 8 samples/libraries please use a 96 well PCR plate with samples loaded in columns.
  • We cannot accept samples/libraries/data prior to the receipt of a sample submission form.

STEP 3 - PROJECT STARTS

  • QC: Your samples/libraries/data will be Quality Checked (QC)
  • Invoice: You will be invoiced according to the agreed quotation or any approved changes to your project
  • Result: You will receive results once your samples/libraries/data have been processed and the invoice has been paid

Pre-prepared libraries/pools submission

Quantify libraries by qPCR and adjust the concentration of library or pool to 4-10nM by using Tris-Cl 10mM, pH 8.5 with 0.1% Tween 20.  

Libraries or pools must be submitted in clearly labelled tubes, if more than 8 libraries or pools are submitted please arrange them into a 96 well PCR plate.

Single Library per Lane:

Submit 15 uL of 4-10 nM library. 

More than one library per lane(s):

Submit 30 uL of 4-10 nM final pool per lane of sequencing requested.

Prior to pooling barcoded libraries, it is essential to normalize the molar concentration of the libraries to ensure that an equal number of reads is generated for each library (using KAPA Library Quantification Kits.) Pool equimolar concentrations of uniquely-indexed libraries and submit 30 uL of the pool per lane of sequencing requested.

*Note: If the library pool concentration is below the submission concentrations mentioned above, contact IGF Facility for further instructions and advice. 

Custom Sequencing Primers:

If using custom sequencing primers, these must be submitted along with samples.

  • Aliquots must be at 100 uM in at least 20 ul
  • Primers must be diluted in EB or DI Water
  • Must be in a Low-bind tube and clearly labeled 

 Best Practices to deal with Illumina HiSeq 4000 Index mis-assignment: 

Relatively high levels of index mis-assignment (commonly known as “index hopping”) have been reported on HiSeq 4000 platform (Sinha et al). Libraries with higher levels of free adaptors will see higher levels of “index hopping”. The free adaptors have the potential to prime and extend library molecules in the same lane during the clustering step. This can result in mis-assignment of reads through index swapping and causing errors in demultiplexing data, as reads from one sample have the potential to end up in the FASTQ files of a different sample.  

A few suggested mitigation strategies to reduce index swaps are listed below: 

  • Remove free adaptors using an extra bead clean-up of each individual library before pooling.
  • Use dual indexing strategies with unique barcodes on both ends. (Swapping would have to occur at both ends for read mis-assignment to occur) (unique i5 and i7 indexes)
  • Store libraries individually at -20°C
  • Pool libraries prior to sequencing 

IMPORTANT: 

- All libraries will be stored by the IGF Facility for three months and then disposed of.

- Please contact us if you wish to collect your library after sequencing. We recommend submission of only an aliquote of your total library prep.

- Libraries received without the appropriate and correct paperwork or poorly labeled, will delay the initiation of your project, risk the safety of your samples and incur additional charges.

RNA sample submission

1.    RNA Extraction:

Total RNA can be extracted using common RNA-extraction kit such as Qiagen RNAeasy kits, Zymo kits (Direct-zol or Quick-RNA miniprep kits) or Invitrogen TRIzol. The use of phenol 

2.    DNase Treatment: 

DNase I treatment needs to be included in the RNA-extraction in order to eliminate the background genomic DNA contamination. Zymo include DNase in their extraction kits or DNase should be purchased separately and added in extraction (TURBO DNase, ThermoFisher). 

3.    RNA Quality and Volume:

  • Provide RNA in minimum 13uL RNase-free water (not in TE or other buffers).
  • Our current protocol requires an input amount of 10 ng–1 µg total RNA quantified by Qubit Fluorometer. However, we recommend to provide higher amounts (min 200ng).
  • High-quality RNA is essential for successful RNA-Seq experiments. High quality RNA shows a 28S rRNA band at 4.5 kb of twice the intensity of the 18S rRNA band at 1.9 kb. RNA with a RIN value above 7.
  • If high quality RNA is not available, our Facility has access to specific library preparation kits that are designed for partially-degraded and FFPE samples with low RIN values. However, the effect of degraded RNA on the sequencing results should be carefully considered.

 4.    RNA storage:

RNase-free conditions must be used in handling the RNA samples. In order to minimize the freeze and thaw cycles of your RNA, please make sure you have our barcoded tubes/plates before RNA-extraction. Store your extracted RNA directly in the provided tubes/plates and transfer a 3uL aliquot of your samples in separate PCR strip tubes to be used for the initial QC in our Facility. Store both barcoded tubes and QC aliquots at -80C.

 5.   We strongly recommend for your RNA samples to be delivered in Dry ice.

 7.   Samples must be submitted in clearly labelled tubes, if more than 8 samples are submitted please arrange them into a 96 well PCR plate in columns.

 8.   Important:

Following the QC of your RNA samples, we will proceed with samples that meet the minimum requirements for the chosen kit. If any samples fails our QC, we'll contact you and give you the option to proceed, remove the sample(s) from the batch for library prep or consider alternative library construction methods. The project and final invoice will be changed accordingly.

Best Practices to deal with Illumina HiSeq 4000 Index mis-assignment: 

Relatively high levels of index mis-assignment (commonly known as “index hopping”) have been reported on HiSeq 4000 platform (Sinha et al). Libraries with higher levels of free adaptors will see higher levels of “index hopping”. The free adaptors have the potential to prime and extend library molecules in the same lane during the clustering step. This can result in mis-assignment of reads through index swapping and causing errors in demultiplexing data, as reads from one sample have the potential to end up in the FASTQ files of a different sample. By using best laboratory practise for cleanup and library storage, we have recently measured this problem to affect less than 1 or 2% of the reads in our hands. For projects requiring high level of confidence for rare variants (e.g. cancer sample analysis) we use a custom dual indexing system. This strategy assignes unique barcodes on each end of libraries. The barcodes are not shared by any onther sample making mis assignment virtually impossible.

DNA sample submission

1. DNA Extraction:

Genomic DNA should be purified using a column-based purification protocol such as the Qiagen DNeasy kit, or one of the DNA purification kits from Zymo Research (Quick-gDNA MiniPrep, Quick-gDNA Blood MiniPrep, or ZR Genomic DNA-Tissue MiniPrep). We recommend elution of the DNA in DNase-free water or tris buffer that does not have EDTA as it may interfere with enzyme activities during the library preparation.

2. RNase Treatment:

RNase treatment needs to be included in the DNA-extraction in order to eliminate the background RNA contamination. 

3. DNA Quality CHeck:

Genomic DNA should be provided in DNase-free water as high molecular weight DNA with no RNA contamination. If samples have been exposed to phenol or other organic solvents, they should be run through a Qiagen cleanup column prior to submission to avoid contaminants that may inhibit the activities of enzymes used in the Illumina library preparation protocols.

 4.    Volume and concentration requirements:

 

 5.    DNA storage recommendations:

 6.   You can submit your DNA samples on ice.

 7.   Samples must be submitted in clearly labelled tubes, if more than 8 samples are submitted please arrange them into a 96 well PCR plate in columns.

 8.   Important:

Following the QC of your DNA samples, we will proceed with samples that meet the minimum requirements for the chosen kit. If any samples fails our QC, we'll contact you and give you the option to proceed, remove the sample(s) from the batch for library prep or consider alternative library construction methods. The project and final invoice will be changed accordingly.

ChIP sample submission

We can only process material which has been already immunoprecipitate by the laboratory requesting service

1.    Chromatin fragmentation done by you:

We recommend optimising your chromatin fragmentation conditions before performing immunoprecipitation in order to maximise the amount of material in the range of 50 to 350bp.

2.    Quality check done by you:

  • A test for enrichment can be performed by qPCR for reagions expected to be bound by the protein of interest. Beware that if chromatine fragmentation is suboptimal and produces predominantly large fragments, the qPCR enrichment test may still be positive but sequencing data will possibly fail to reveal the same enrichment. This is because large fragments are very inefficient or comletely fail to generate good quality clusters and will not contribute to the set of sequences.
  • Quantification should be done using Qubit or picogreen as nanodrop overestimates the amount of material present. 

3.    Immuno precipitated DNA concentrtation and Volume:

We require 10ng of material. Samples should be in a volume of no more than 20μl of ultrapure water. Input samples may need dilution, however, please do not dilute
ChIP samples as this may make them undetectable when we perform our QC of your samples.

4.    DNA storage recommendations:

 5.   You can submit your DNA samples on ice.

 6.   Samples must be submitted in clearly labelled tubes, if more than 8 samples are submitted please arrange them into a 96 well PCR plate in columns.

 7.   Important:

Immunoprecipitated samples are often very diluted and may be undetectable when we perform our QC, however, we will proceed with library prep of all samples. Following the QC of the libraries, if any samples fails our QC, we'll contact you and give you the option to proceed or remove the sample(s) from the batch for sequencing. In either case we will charge for library construction.

 More detailed information regarding experimental design and analysis is provided by Landt et al.

Cells sample submission

under construction

 


Best Practices to deal with Illumina HiSeq 4000 Index mis-assignment: 

Relatively high levels of index mis-assignment (commonly known as “index hopping”) have been reported on HiSeq 4000 platform (Sinha et al). Libraries with higher levels of free adaptors will see higher levels of “index hopping”. The free adaptors have the potential to prime and extend library molecules in the same lane during the clustering step. This can result in mis-assignment of reads through index swapping and causing errors in demultiplexing data, as reads from one sample have the potential to end up in the FASTQ files of a different sample. In our hands, by using best laboratory practise for cleanup and library storage, we have found that less than 1 or 2% of the reads are mis-assigned.

However, for projects requiring high level of confidence for rare variants (e.g. cancer sample analysis) we use a custom dual indexing system. This strategy assignes unique barcodes on each end of each libraries. The barcodes are not shared by any onther sample making mis assignment virtually impossible.