Originally posted by ETHANol
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On a related note; I have a bunch of indexes (The Sanger 96-plex ones) that I'd like to use for low plexing too (4plex). These indexes are 8 bases long. Is there anything stopping just reading the first 6 bases (as per standard illumina indexing) on the GAIIx/HiSeq as long as there is AC/GT balance at all 6 positions? I only want to do this on one lane, so don't need to read 8 cycles on the other 7 lanes.
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Originally posted by ETHANol View Posthttp://www.plosone.org/article/info%...l.pone.0016607
With a 5' index you have the invariant T required for adapter ligation in all libraries. I guess it doesn't cause too much of a problem because people use this strategy, but it is something to think about nonetheless. Has this caused problems for anyone?
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Originally posted by ETHANol View Posthttp://www.plosone.org/article/info%...l.pone.0016607
With a 5' index you have the invariant T required for adapter ligation in all libraries. I guess it doesn't cause too much of a problem because people use this strategy, but it is something to think about nonetheless. Has this caused problems for anyone?
You mean the T for the T-A ligation right? No I don't think it's a problem because that T (or actually its complement A) anneals to the last base of the sequencing primer so essentially it's not part of the read
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Originally posted by kentk View PostThanks guys. Yes I was planning to introduce a 5' index. Being able to multiplex only at multiples of 4 isn't a problem. Just need to multiplex into the hundreds.
I think I've read the same post by pmiguel mentioning index reads should always contain a A/C and G/T at each position that is why I was curious why all bases should be in equal proportion.
For the index read, A/C vs. G/T is usually sufficient to discriminate between a small number of barcodes.
Harold
Harold
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Massively parallel DNA sequencing is capable of sequencing tens of millions of DNA fragments at the same time. However, sequence bias in the initial cycles, which are used to determine the coordinates of individual clusters, causes a loss of fidelity in cluster identification on Illumina Genome Analysers. This can result in a significant reduction in the numbers of clusters that can be analysed. Such low sample diversity is an intrinsic problem of sequencing libraries that are generated by restriction enzyme digestion, such as e4C-seq or reduced-representation libraries. Similarly, this problem can also arise through the combined sequencing of barcoded, multiplexed libraries. We describe a procedure to defer the mapping of cluster coordinates until low-diversity sequences have been passed. This simple procedure can recover substantial amounts of next generation sequencing data that would otherwise be lost.
With a 5' index you have the invariant T required for adapter ligation in all libraries. I guess it doesn't cause too much of a problem because people use this strategy, but it is something to think about nonetheless. Has this caused problems for anyone?
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Originally posted by ETHANol View PostHESmith, Thanks for the correction. I'm curious here. How do you determine that the indices are called incorrectly? How do I go about performing QC on the index read?
You can use SAV or HCS to visualize the Q-scores for the index cycles. They are usually a bit lower than read one; if they're a lot lower, be concerned. A high fraction (>3-4%) of reads in the Undetermined directory is another indication of poor index reads.
Harold
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Thanks guys. Yes I was planning to introduce a 5' index. Being able to multiplex only at multiples of 4 isn't a problem. Just need to multiplex into the hundreds.
I think I've read the same post by pmiguel mentioning index reads should always contain a A/C and G/T at each position that is why I was curious why all bases should be in equal proportion.
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It's funny, I found this on the internet some time ago and follow it but Illumina hasn't followed up on it so I just assumed it wasn't a problem. Apparently, it can be. Which leads one to ask, why is this not mentioned in any of the library preparation manuals.
I think pmiguel has said that base balanced base composition for the index read is important on the HiScan.
1. Some sequencing experiments require the use of fewer than 12 index sequences in a lane with a high cluster density. In such cases, select indexes carefully to ensure optimum base calling and demultiplexing by having different bases at each cycle of the index read. Illumina recommends the following sets of indexes for low-level pooling experiments.
Pool of 2 samples:
• Index #6 GCCAAT • Index #12 CTTGTA
Pool of 3 samples:
• Index #4 TGACCA • Index #6 GCCAAT • Index #12 CTTGTA
Pool of 6 samples: • Index #2 CGATGT • Index #4 TGACCA • Index #5 ACAGTG • Index #6 GCCAAT • Index #7 CAGATC • Index #12 CTTGTA
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HESmith, Thanks for the correction. I'm curious here. How do you determine that the indices are called incorrectly? How do I go about performing QC on the index read?
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Actually, ETHANol's statement is not 100% accurate. On the HiSeq, high cluster densities (900-1000K) have a more deleterious effect on index reads than inserts. We've had several flow cells with good cluster calling (80-90% PF) and high quality scores (mean ~38), yet fewer than 50% of the indices were called accurately. In some cases, pseudotiles at the inflow side (which contain higher cluster densities) have completely dropped out (i.e., no basecalling) during the index read after producing high-quality insert reads. The problem can be mitigated by balancing the ratio of index bases that are excited by the same laser (A/C or G/T).
If your second index is at the start of read one, then you absolutely have to use all four bases in roughly equal proportions for the first four cycles (which is when cluster calling occurs).
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On the HiSeq, you need balance nucleotide composition at the beginning of the sequencing read but not the barcode read. Otherwise you could only multiplex in multiples of four. Which was one of the drawbacks of putting the barcode at the beginning of the sequencing read. Maybe the MiSeq is more picky about the barcode read, I don't know.
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Yes I should maximize hamming distance.
But for example I have 4 indexes...
5' ATGCAT
5' TGAACG
5' GCTGTC
5' AGCTGC
An Illumina representative mentioned that I can't use that index set because the first, second and last bases will not have all of the four bases. So one the flowcell for base 1, I'll have signals for A, T, G clusters but not for C so our machine (MiSeq) will trash that cycle. Well this is what I understood from our conversation.
Any thoughts?
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Not really sure if I am getting what you are saying but it's best to keep all the hamming distance >1 for all the barcodes.
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