Experience exchange seminar

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[править] About the seminar

We are three independent groups dealing with different aspects of NGS. Every two weeks we meet together for experience exchange. Each person tells the others something new, interesting and/or important about his/her area of work. It might be a hint on some improvement, comment to the protocol, information about new kit, description of a problem arisen in the work, etc. If there is nothing new in the own work, it is necessary to find useful information elsewhere (internet, journals, etc.)

Summary of each seminar is placed here. Problems mentioned are tracked and their status updated directly on this page. From time to time, comments to the protocols and useful tips and tricks are transferred to the page of the corresponding protocol.


[править] Seminar 28.10.2011

Research news

  • Interesting paper came out in Nature Biotechnology -- “Efficient targeted resequencing of human germline and cancer genomes by oligonucleotide-selective sequencing” (Myllykangas et al. 2011 Nature Biotechnology, Vol.29:11, pp 1024-1029).

The authors suggest to perform library enrichment directly on the flowcell. Enrichment probes are first loaded onto the flowcell, and then used to fish out library molecules containing inserts from the region of interest. Described is a proof-of-principle experiment. 90% of obtained reads matched target regions, which is good – with SureSelect ~85% of reads match target region. However, only ~2 mln reads is obtained from a single lane of GAII (the normal number of clusters for that version of GAII was ~10 mln).


RNA-seq library preparation

  • TruSeq mRNA kit is compatible with our strand-specific uridine protocol.
  • DNA/RNA purification after first strand synthesis: home-made G-50columns work better, than commercial columns (###). 50% cDNA output with commercial (Agnes).
  • It was reported about comparison of 3 commercial PolyA selection kits (###):TruSeq, Ribominus and Nugen Ovation Kit.
- TruSeq (using oligo dT magnetic beads) gave better results than Ribominus: less rRNA, intronic and intergenic regions.
- in case small amounts of starting material is used, it is appropriate to use Nugen Ovation Kit (works good up to 500pg total RNA).


Sequencers

  • Third Generation Sequencer from Pacific Biosciences is installed at the Berlin Institute for Medical Systems Biology (BIMSB) of the Max Delbrück Center for Molecular Medicine (MDC) Berlin-Buch. The BIMSB is the first academic research institution in Continental Europe to use this sequencer for research.
  • HiSeq
- guideline values of intensities (in case lower values → rehybridisation):
previously: A int (15-30.000), C int (20-40.000), G int (5-15.000), T int (15-30.000)
new SBS Kit v3: new values are accepted: G int about 5.000 is OK; T int between 6-8.000 was also accepted by TechSupport
- ???Test hybridisation result: G int raised by about 2.500
- clustered FC maybe stored for 10 days; for longer storage, it is recommended to repeat rehybridisation prior to sequencing run
- saved runs:
Read 2 resynthesis failed: int dropped down to 0 → rehybridisation using special protocol was performed
Peltier element would not work (no temperature profile, images identical in all cycles) → stop run, rehybridisation, starting on the other HiSeq.


Lab equipment

  • Trial of Beckman Coulter Biomek 2000 Robot (Lesha)

Biomek was tested for cDNA synthesis starting from 10 to 180ng polyA RNA.

- higher yield than manually for small amounts – 80% (10ng polyA → 8ng cDNA)
- the same yield as manually for large amounts
- RNA fragmentation was performed in the machine


NA staining

  • Fluorescent dyes (Masha)
- EtBr – ds and ss NA staining
- Sybr I – dsNA staining
- Sybr II – ssNA staining
- sensitivity: EtBR < Sybr I and II < SybrGold
  • SybrSafe is produced as EtBr exchange: claimed to be safe for worker but much more expensive, about 5€ per gel


[править] Seminar 14.10.2011

Problems

  • ??? Bone marrow patient samples: final RNA-Seq libraries show 2 peaks on qPCR melting curves. Libraries prepared in parallel from other tissues (same amounts, procedure) show single major peak, as usual. (Kerstin).
status 28.10.11 - libraries are sequenced, analysis in progress
  • ??? How to calculate from the library complexity amount of sequences required (Mathias)?
  • ??? DNA shearing: the bigger size is required, the longer smear is obtained (for Covaris and UTR). Fragments of bigger size have less shearing accuracy (Kerstin)
status 28.10.11 - Shearing procedure is being selected
  • Problems with genomic DNA seq. libraries preparation in 1000 Genome project. Probably Ph/Cl before processing DNA would not hurt? (Agnes)
status 28.10.11 - Ph/Cl before processing DNA improved the situation
  • New mate-pair library protocol (ABI) worked on test DNA samples from mouse liver but failed for the 1000 Genome samples.
- genomic DNA coming should be better Ph/Cl purified – maybe that was the reason
status 28.10.11 - Ph/Cl before processing DNA improved the situation
- ??? according to ABI input DNA should be between 5 and 10 µg (for a insert size > 1 Kb and for a sequencing read with coverage > 20x) and for circularisation they take 500ng, expecting 15-25% circularised molecules (~100ng) ---- in our previous experience, minimum amount of circularised DNA to get a decent library (~109 complexity) was 300ng.
- ??? according to ABI expected yield for a final library schould be 25 ng (for a FC 7-8 ng need for the 5500 Solid and 5 ng - for a full slide for Solid 4). But this means only 8x107 in terms of complexity (calculated for 10 cycles for final amplification and 300bp fragments sizes)


Lab equipment

  • Trial of Beckman Coulter Biomek 2000 Robot (Lesha):

(http://www.selectscience.net/products/biomek-2000-laboratory-automation-workstation/?prodID=83868) Nice feature of this robot is flexibility: users can use ready company made programs as well as write their own programs.

Biomek was tested for mRNA purification. Input: 20, 10, 5, 2.5µg total RNA; output 1-2% polyA – like for the manual procedure. The process overall takes 2h with about 15 min of handwork. Overall impression: good. There was a problem: during bead washing steps, beads are held on a magnet for 2 min.; after that just shaking of the plate is not enough to resuspend the beads in the solution, pipetting was required.


NGS web resources

NGS community web site with an active forum for all aspects of next-generation genomics.


Genomic DNA library preparation

  • New Mate-Pair library kit from ABI (Simon).

Uses AMPure Beads for purification steps i.e. there is a possibility to work with a pipetting robot like Biomek. Gives 15-25% circularisation output comparing to 10% of the previous protocol. (Simon)

  • WGA-NGS Kit (Rubicon Genomics): 1300 Euros for 10 reactions

WGA – whole genome amplification. Kit for making libraries from < 10ng of genomic DNA. Material is first amplified using cyclic random primer extension. Random primers have known overhangs which are used for PCR amplification. Roughly from 10ng one gets ~1000ng of purified, fragmented (200-600bp) DNA. Representation of different regions in the amplified material varies within 8 times.

Amplified material may be processed as a standard DNA in Illumina protocol (end-repair, A-tailing, etc.) or Illumina compatible adapter sequences may be introduced during one more round of PCR amplification. Special sequencing primers are required for the library. (Vera)

Sequencers

  • MiSeq (Alex and Jeaninne)

MiSeq is a new sequencer from Illumina which we will get soon. It combines the cluster generation/amplification and the sequencing in one instrument. The flowcell has just one lane so the sequencing goes very fast. E.g. a paired-end run with 2x 100 cycles takes about 19 hours for amplification and sequencing in the machine. (without the data analysis) The chemistry comes in single-use cartiges. There are two different kits available: 1. 300 cycle kit - 323 cycles (2x 8 cycles for indexing) 2. 50 cycle kit - 73 cycles (2x 8 cycles for indexing)



[править] Seminar 30.09.2011

Problems

  • ??? Genomic DNA shearing to 350-450bp, huge losses during primary size selection. Covaris and UTR200 give too large a smear and there is not much DNA in the desired region.
Status 27.10.11: checking different sonicators (UTR, Bioruptor)at different conditions.


RNA isolation

  • Hint: by isolating RNA from fresh bone marrow - do not freeze lymphocytes.


RNA-Seq library preparation

  • To obtain an RNA-Seq library of ~108 complexity (for the ready, amplified libraries), there should be ≥ 10ng of ds cDNA and, correspondingly, ≥ 2µg of total RNA (cDNA shearing protocol).
  • RIN does not matter when making library – we got libraries of ~109 complexity even with RIN ~7.
  • ??? There is an interesting kit from Epicentre for RNA library preparation - ScriptSeq™ mRNA-Seq Library Preparation Kit ( http://www.epibio.com/item.asp?ID=578 ). Random primers for the first and second strand cDNA synthesis contain different tails, providing strand-specific information. Kerstin has ordered this kit and will try it out. Not really clear what are the advantages.
  • Chemical fragmentation of RNA:

for 2x100bp reads: 3 min at 60°C (obtained range: 84-1126bp, max at 243bp) for 2x50bp reads we used: 5 min at 70°C (obtained range: 70-380bp, max at 131bp)

  • ??? Glycogen inhibits cDNA synthesis for small (<3ng of polyA RNA) amounts of starting material in ~2 times. Actually this should not influence our standard RNA-Seq library preparation. Kerstin suggested to use linear polyacrylamide (LPAA)
  • It is possible to synthesize cDNA from down to 100pg of mRNA. Output can be measured by qPCR with primers for specific genes (for example G3PDH)


Genomic DNA library preparation

- kit works; from 50 ng of genomic DNA she got library with complexity 2x109 (as for the standard protocol)
- problems: gave too small insert sizes for 2x100 PE library: maximum intensity ~250 bp with adaptors (adaptors length 135 bp) though with HMW buffer, total range 150-800 with adaptors;
- when the problem with sizes is fixed we could test the kit on TG samples
  • ??? New Mate-Pair library kit is available from ABI. Differences to the previous one: no internal adapter during circularization, instead - long sticky ends blocked by oligos till circularization; A tailing before adapter ligation. Simon will try it.


Lab equipment

  • ??? new thermotransfer printer (Brady, ####, $826, web-site, consumables) will arrive soon
- available through internet
- labels withstand nitrogen freezing
- preconfigured labels, 1 and 2 dimensional barcodes
- compatible with LIMS software
Status 28.20.11 - printer has arrived


Sequencers

  • HiSeq: new kit available for manual removal of bubbles from the syringes (Spare Kit, FSE Ref: 15013841S). Kit and the two wash-FCs are must-bes.
  • Working on cBot under administrative rights - allows to skip some processes.
  • New sequencer MaxSeq is available on the market (AZCO Biotech,Inc), successor of the Polonator:
- up to 100bp reads, SR and PE
- is claimed: up to 100GB a run, at 30% of the HiSeq price
- works with beads (like SOLiDs) and rolonies (RCA products)
- sequencing by ligation and by synthesis
- instrument costs ~$300000


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