No sample QC is done so it’s very important that you make sure the above sample requirements are fulfilled. Tagmentation cannot add an adapter directly to the distal end of a fragment, so a drop in sequencing coverage of around 50 bp from each distal end is expected. The PCR amplicon must be larger than 150 bp, as shorter amplicons can be lost during the library cleanup step. This does not mean that FFPE samples cannot be attempted, but that failed libraries originating from this sample type are not eligible for replacement or troubleshooting. Special sample types FFPEįFPE or degraded DNA as input for this this protocol is not supported by the manufacturer. So the compromise is that you are responsible that your samples are provided in the proper range of concentrations the read variability may be greater compared to when we individually quantify and dilute libraries before pooling them, but the cost will be lower. At the same time we won’t make any guarantees regarding the variability in data yield among the samples. We then pool the libraries by equal volume relying on the built-in normalisation. The concept of user QC is that you provide your samples within the specified range of input (100–500 ng) and libraries are prepared without us doing any sample QC. Two last positions in the last column MUST be left empty for the controls. This means we can only accept sample batches of 94. By only processing full plates (including two control samples) the reagent dead volume and consumable cost is minimised. User QCīecause fragmentation is enzymatic and there is a degree of built-in sample normalisation in this chemistry, we can skip Covaris shearing and quantification steps that add time and cost to the library preparation. We currently have access to 384 (combinatorial) dual indexes which means that is the highest level of multiplexing possible. These substances can interfere with the Nextera tagmentation reaction and result in unexpected library insert sizes. Make sure that the DNA sample does not contain more than 1 mM EDTA and is free of organic contaminants, such as phenol and ethanol. NGI will include 2 controls in the prep, please leave the last two positions in the last column empty To take advantage of the built-in normalisation, the input must be above 100 ng.ġ0 mM Tris, pH 8–8.5 or similar (e.g. For small genomes, the DNA input amount can be reduced to as low as 1 ng (requiring more PCR cycles). For human DNA samples and other large complex genomes, the recommended DNA input is between 100–500 ng. The Illumina DNA protocol is compatible with DNA inputs ranging from 1-500 ng.

With this method of normalisation, we typically see a variation in sequence reads of 20% CV among samples in a pool.

Because of how the enzymatic fragmentation works, we can only offer this method with 350 bp insert sizes.

Low cost library prep option, only available in batches of 94 samples.This makes it a comparatively fast protocol which can produce libraries with a consistent yield and fragment size distribution from a wide DNA input range. The Illumina DNA library prep is based on the bead-linked transposome (BLT) complex which enables both fragmentation of genomic DNA and adding adaptor sequence to those fragments in a single step. Sequence: CTGTCTCTTATACACATCT Type: regular 3' Length: 19 Trimmed: 1459 times.Low cost library preparation option for gDNA based on bead-linked transposase. ResultsĪs a result, here is the number of sequences trimmed by cutadapt: Total read pairs processed: 5,441,659 The difficult part in this procedure is to chose a number for the –overlap parameter, which defines the minimum overlap between a read and an adapter. Script to trim Nextera Transposase Sequence (length of 19 nucleotides): adapter=' CTGTCTCTTATACACATCT'Ĭutadapt -a $_R2.fastq.gz In this post, we will use the following version of cutadapt: cutadapt -version 2.3 Scripts used However, the adapter sequences recommended by Illumina for Nextera XT and TruSeq are longer than the ones used by FastQC: Library preparation kitĪdapter sequences trimmed by cutadapt Tool used Adapter sequences used by FastQCīy default, FastQC searches for several adapter sequences in each library, including those two: Adapter name But, if the insert size is smaller than the read length, adapter sequences can be found in the reads at the 3′ end.

What are adapter sequences?Īfter library preparation, adapter sequences are present on both 5′ and 3′ ends of the DNA fragments and are not supposed to be sequenced. In this tutorial, we will cover how to trim adapters such as the Nextera Transposase Sequence and the Illumina Universal Adapter with cutadapt.

Nextera adapter sequences