De-multiplexing, carryover from sample with highest reads?
Hello,
I use in house indexes (5nt) to tag my amplicons and pool 10 such samples and subject the pooled "sample" to library prep on TruSeq without fragmentation.
After demultiplexing for Illumina indexes on Miseq, FASTX barcode splitter is used to de-multiplex the samples to the original "samples". These reads are then aligned with Bowtie and the result we need is a proportion of reads aligned aganist a particular reference sequence.
When the final alignment results are observed, in all samples there is a carry over of some reads from the sample with the highest number of reads to the rest of the samples in the same 10 sample pool.
I checked to see if this is associated with any particular pattern of the in house barcodes, it is not. In all the samples, it is the reads from the sample with "highest number of reads" that are also found in other samples.
Has anyone else experienced something similar in sample pooling and is there any other software that has a more stringent algorythm for barcode splitting.
Not sure if the post is confusing
I use in house indexes (5nt) to tag my amplicons and pool 10 such samples and subject the pooled "sample" to library prep on TruSeq without fragmentation.
After demultiplexing for Illumina indexes on Miseq, FASTX barcode splitter is used to de-multiplex the samples to the original "samples". These reads are then aligned with Bowtie and the result we need is a proportion of reads aligned aganist a particular reference sequence.
When the final alignment results are observed, in all samples there is a carry over of some reads from the sample with the highest number of reads to the rest of the samples in the same 10 sample pool.
I checked to see if this is associated with any particular pattern of the in house barcodes, it is not. In all the samples, it is the reads from the sample with "highest number of reads" that are also found in other samples.
Has anyone else experienced something similar in sample pooling and is there any other software that has a more stringent algorythm for barcode splitting.
Not sure if the post is confusing
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