Using Unique Molecular IDs with NEBNext Direct®  - Data Usage Guideline Page

A downloadable pdf version of this guideline is available here.

In order to reap the benefits of the Unique Molecular IDs (UMIs) with NEBNext Direct libraries it is necessary to:

  1. Insert the UMI sequences into your SAM/BAM files
  2. Mark or remove duplicate reads with a program that is UMI-aware

The following two sections contain recommendations for both steps.

Inserting UMIs into SAM/BAM Files

The simplest way to introduce UMIs into an existing pipeline is to use a tool that will insert UMIs into pre-existing SAM/BAM files.   For this purpose we recommend using the AnnotateBamWithUmis tool from the fgbio package, available from:

Follow the instructions on the website to download and unpack the package. Following that, UMIs are added to a BAM using:
java -Xmx4g -jar fgbio.jar AnnotateBamWithUmis \
    -i in.bam -f umi.fastq -o out.bam 

By default this will create a new file, out.bam, identical the the file in.bam, but with an additional tag RX that contains the UMI sequence read for each record.  To override the tag name used to store the UMI, use the -t option:
java -Xmx4g -jar fgbio.jar AnnotateBamWithUmis \
    -i in.bam -f umi.fastq -o out.bam -t XX    

Finally full help and usage information is avaiable by runing:
java -Xmx4g -jar fgbio.jar AnnotateBamWithUmis --help 

Note: if your pipeline starts with Illumina BCL files instead of Fastq files you may wish to explore using IlluminaBasecallsToSam from the Picard package which is capable of converting from BCL to BAM, demultiplexing, and inserting UMI tags in a single pass.  For more information see:

Marking Duplicates with UMIs

For marking PCR duplicates within a library we recommend using MarkDuplicates from the Picard suite of tools.  Duplicating marking with UMIs is an optional feature, and must be turned on explicitly.  To mark duplicates in a BAM file containing UMIs run:

java -Xmx4g -jar picard.jar MarkDuplicates \
  INPUT=in.bam \
  OUTPUT=out.bam \
  METRICS_FILE=metrics.txt \

If a different tag name was provided earlier, substitute that tag name for RX here.

For further details and the full set of available options to MarkDuplicates refer to the online documentation: