Dual-index sequencing of V4 amplicons on Miseq

I would like to sequence 384 sludge samples in one of the two libraries (of a lane) on Miseq using bacteria and archaea V4 region universal priemr set: ArBa515F: GTGCCAGCMGCCGCGGTAA and ArBa806R: GGACTACVSGGGTATCTAAT, following the method propose by Kozich et al., 2013 of the mothur Group. The foward priemr ArBa515F is exctly the same as the one used in the paper, while the reverse primer ArBa806R is slightly different from the 806R used for V4 region of bacteria (see below). Does it still work if I use the same Barcode, padR (AGTCAGTCAG), linkF (GT), linkR (CC) as those in Kozich et al., 2013?? Or changes are needed in the Link or/and pad sequences???

806R: 5’-GGA CTA CHV GGG TWT CTA AT-3’
ArBa806R: 5’-GGA CTA CNS GGG TMT CTA AT-3’

Below is a sequence list of (I) my priemrs, (II) two sequencing primers, and (III) two index sequencing primers to be synthesized, and my gratitudes for any comments on their effectiveneess or correctness:
(1) 5’ Illumina adapter|Barcode|padF|linkF|Forward Primer: AATGATACGGCGACCACCGAGATCTACAC ATCGTACG TATGGTAATT GT GTGCCAGCMGCCGCGGTAA
(2) 5’ Illumina adapter|Barcode|padF|linkF|Reverse Primer: CAAGCAGAAGACGGCATACGAGAT AACTCTCG AGTCAGTCAG CC GGACTACVSGGGTATCTAAT
(3) Read 1 sequencing primer (padF|linkF|Forward primer): TATGGTAATT GT GTGCCAGCMGCCGCGGTAA
(4) Read 2 sequencing primer (padF|linkF|Reverse primer): AGTCAGTCAG CC GGACTACVSGGGTATCTAAT
(5) First (Read2) index sequencing primer (reverse complement of (4)): ATTAGATACCCSBGTAGTCC GG CTGACTGACT
(6) Second (Read 1) index sequencing primer: AATGATACGGCGACCACCGAGATCTACAC (I do not konw how this sequence is determeind)

Another question is that, can I combine together amplicons of V4-bacteria and V4-universal into the same library when applies the above Dual-index sequencing strategy??
If yes, how to coordinate the difference in the 6 seqeucnes in the above list??? I would like first to test the effectiveness of the above sequence list before applying them to a lane of samples.

Thanks very in advance for any of your insights into the above two questions!!

Does it still work if I use the same Barcode, padR (AGTCAGTCAG), linkF (GT), linkR (CC) as those in Kozich et al., 2013?? Or changes are needed in the Link or/and pad sequences???

It should work without having to change the barcode and pad. The linker is a 2 bp anti-sense sequence to anything existing in the database. I’m not sure how important the exact sequence of the linker is, but it makes us feel good about ourselves.

Another question is that, can I combine together amplicons of V4-bacteria and V4-universal into the same library when applies the above Dual-index sequencing strategy??
If yes, how to coordinate the difference in the 6 seqeucnes in the above list??? I would like first to test the effectiveness of the above sequence list before applying them to a lane of samples.

I would probably assign them different barcodes/indices so that it is easier to fish them apart later.

Pat

Thanks, Pat! I will use different barcodes.
But if I combine together amplicons of V4-bacteria and V4-universal into the same library, there are two pairs of primers, one for V4-bacteria, and the other for V4-universal. Then the question comes that how can I coordinate the difference in the forward and reverse primers in the 6 synthesized seqeucnes below? Do I need to synthesize independently two sets of sequences using the two pairs of primers and finally combined them in a library for sequencing on Miseq? Your comments are highly appreciated!

(1) 5’ Illumina adapter|Barcode|padF|linkF|Forward Primer: AATGATACGGCGACCACCGAGATCTACAC ATCGTACG TATGGTAATT GT GTGCCAGCMGCCGCGGTAA
(2) 5’ Illumina adapter|Barcode|padF|linkF|Reverse Primer: CAAGCAGAAGACGGCATACGAGAT AACTCTCG AGTCAGTCAG CC GGACTACVSGGGTATCTAAT
(3) Read 1 sequencing primer (padF|linkF|Forward primer): TATGGTAATT GT GTGCCAGCMGCCGCGGTAA
(4) Read 2 sequencing primer (padF|linkF|Reverse primer): AGTCAGTCAG CC GGACTACVSGGGTATCTAAT
(5) First (Read2) index sequencing primer (reverse complement of (4)): ATTAGATACCCSBGTAGTCC GG CTGACTGACT
(6) Second (Read 1) index sequencing primer: AATGATACGGCGACCACCGAGATCTACAC (I do not konw how this sequence is determeind)

Thanks, Pat! I will use different barcodes.But if I combine together amplicons of V4-bacteria and V4-universal into the same library, there are two pairs of primers, one for V4-bacteria, and the other for V4-universal. Then the question comes that how can I coordinate the difference in the forward and reverse primers in the 6 synthesized seqeucnes below? Do I need to synthesize independently two sets of sequences using the two pairs of primers and finally combined them in a library for sequencing on Miseq? Your comments are highly appreciated!

(1) 5’ Illumina adapter|Barcode|padF|linkF|Forward Primer: AATGATACGGCGACCACCGAGATCTACAC ATCGTACG TATGGTAATT GT GTGCCAGCMGCCGCGGTAA
(2) 5’ Illumina adapter|Barcode|padF|linkF|Reverse Primer: CAAGCAGAAGACGGCATACGAGAT AACTCTCG AGTCAGTCAG CC GGACTACVSGGGTATCTAAT
(3) Read 1 sequencing primer (padF|linkF|Forward primer): TATGGTAATT GT GTGCCAGCMGCCGCGGTAA
(4) Read 2 sequencing primer (padF|linkF|Reverse primer): AGTCAGTCAG CC GGACTACVSGGGTATCTAAT
(5) First (Read2) index sequencing primer (reverse complement of (4)): ATTAGATACCCSBGTAGTCC GG CTGACTGACT
(6) Second (Read 1) index sequencing primer: AATGATACGGCGACCACCGAGATCTACAC (I do not konw how this sequence is determeind)

Sorry - I’m not really sure what you’re asking…

Thanks, Pschloss! In the original paper of dual-index sequencing, the authors were using V4 bacteria universal primers for sequencing 384 samples.
My question is that, can I use V4 bacteria universal primers for the first 300 samples and V4 archaeal universal primers for the other 84 samples for amplification, and finally combine them together for sequencing on Miseq?

If the reads are similar in length then you should be fine. I would suggest either using the 250PE kit or the 300PE kit where you only do 250 cycles per read.

Pat