The pipeline

All routines in the pipeline are available in manual and batch variants. Batch variants are designed to automatically handle several input samples with minimal shell scripting glue between analysis steps, this is also the recommended way to use with MIGEC.

List of MIGEC batch routines:

• Checkout-batch
• MIG statistics
• Assemble-batch
• CdrBlast-batch
• FilterCdrBlastResults-batch

If the “barcodes” file is set properly, the entire pipeline can be written as following:

MIGEC="java -Xmx8G -jar MIGEC-$VERSION.jar"
$MIGEC CheckoutBatch -cu barcodes.txt checkout/
$MIGEC Histogram checkout/ histogram/
$MIGEC AssembleBatch -c checkout/ histogram/ assemble/
$MIGEC CdrBlastBatch -R TRB checkout/ assemble/ cdrblast/
$MIGEC FilterCdrBlastResultsBatch cdrblast/ cdrfinal/

Manual usage

List of MIGEC manual routines:

• Checkout-manual
• MIG statistics
• Assemble-manual
• CdrBlast-manual
• FilterCdrBlastResults-manual

An example for a 300bp paired-end MiSeq run of IGH library on a 16Gb RAM Unix server. Such sequencing read length allows complete IGH sequencing, thus mate pairs overlap. First barcodes.txt should be created containing adapter sequences, see the section below for guidelines. Then, assuming that the corresponding FASTQ files are IGH_SAMPLE_R1.fastq.gz and IGH_SAMPLE_R2.fastq.gz, UMI- and multiplex index-containing adapter is near 5’UTR of V segment (so the CDR3 is in mate#2 after reads are oriented) and NCBI-BLAST+ is installed, run all 5 stages of the pipeline using the following command:

$MIGEC Checkout -cute --overlap barcodes.txt IGH_S1-10_R1.fastq.gz IGH_S1-10_R2.fastq.gz checkout/
$MIGEC Histogram checkout/ histogram/
$MIGEC Assemble -c --mask 0:0 checkout/S1_R12.fastq.gz . assembly/
$MIGEC CdrBlast -R IGH checkout/S1_R12.fastq.gz cdrblast/S1_raw.txt
$MIGEC CdrBlast -a -R IGH assembly/S1_R12.fastq.gz cdrblast/S1_asm.txt
$MIGEC FilterCdrBlastResults cdrblast/S1_asm.txt cdrblast/S1_raw.txt cdrfinal/S1.txt

Note

As dot (.) is reserved by MIGEC to specify empty metadata fields and unused arguments, use ./ in case you want to point to current directory.

Full-length immunoglobulin data analysis

MIGEC can be used as pre-processing tool to assemble full-length consensuses for further post-analysis with HIgBlast tool. Note that due to typically poor quality of MiSEQ 300+300bp reads, the --overlap mode of Checkout routine is not guaranteed to perform well. Instead, we recommend to assemble consensuses first and then perform overlapping using external tools. For example, MiTools merge action can be used with the --same-strand option specified, the latter is critical as assembled consensuses are on the same strand in output in contrast to normal orientation of Illumina reads.

Consensus quality and overlap efficiency can be greatly improved using the --only-first-read option of Histogram and Assemble routines. If set, this option instructs routines to use only the first read that typically has higher quality than the second one. This applies to non-oriented reads and works better for asymmetric sequencing design, e.g. 400+200bp reads.